Prodrugs of methyl hydrogen fumarate, pharmaceutical compositions thereof, and methods of use

ABSTRACT

Prodrugs of methyl hydrogen fumarate, pharmaceutical compositions comprising prodrugs of methyl hydrogen fumarate, and methods of using prodrugs of methyl hydrogen fumarate and pharmaceutical compositions thereof for treating diseases such as psoriasis, asthma, multiple sclerosis, inflammatory bowel disease, and arthritis are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 61/090,163 filed Aug. 19, 2008, whichis incorporated by reference in its entirety.

FIELD

Disclosed herein are prodrugs of methyl hydrogen fumarate,pharmaceutical compositions comprising prodrugs of methyl hydrogenfumarate, and methods of using prodrugs of methyl hydrogen fumarate andpharmaceutical compositions thereof for treating diseases such aspsoriasis, asthma, multiple sclerosis, inflammatory bowel disease, andarthritis.

BACKGROUND

Fumaric acid esters (FAEs) are approved in Germany for the treatment ofpsoriasis, are being evaluated in the United States for the treatment ofpsoriasis and multiple sclerosis, and have been proposed for use intreating a wide range of immunological, autoimmune, and inflammatorydiseases and conditions.

FAEs and other fumaric acid derivatives have been proposed for use intreating a wide-variety of diseases and conditions involvingimmunological, autoimmune, and/or inflammatory processes includingpsoriasis (Joshi and Strebel, WO 1999/49858; U.S. Pat. No. 6,277,882;Mrowietz and Asadullah, Trends Mol Med 2005, 111(1), 43-48; and Yazdiand Mrowietz, Clinics Dermatology 2008, 26, 522-526); asthma and chronicobstructive pulmonary diseases (Joshi et al., WO 2005/023241 and US2007/0027076); cardiac insufficiency including left ventricularinsufficiency, myocardial infarction and angina pectoris (Joshi et al.,WO 2005/023241; Joshi et al., US 2007/0027076); mitochondrial andneurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease, Huntington's disease, retinopathia pigmentosa and mitochondrialencephalomyopathy (Joshi and Strebel, WO 2002/055063, US 2006/0205659,U.S. Pat. No. 6,509,376, U.S. Pat. No. 6,858,750, and U.S. Pat. No.7,157,423); transplantation (Joshi and Strebel, WO 2002/055063, US2006/0205659, U.S. Pat. No. 6,359,003, U.S. Pat. No. 6,509,376, and U.S.Pat. No. 7,157,423; and Lehmann et al., Arch Dermatol Res 2002, 294,399-404); autoimmune diseases (Joshi and Strebel, WO 2002/055063, U.S.Pat. No. 6,509,376, U.S. Pat. No. 7,157,423, and US 2006/0205659)including multiple sclerosis (MS) (Joshi and Strebel, WO 1998/52549 andU.S. Pat. No. 6,436,992; Went and Lieberburg, US 2008/0089896; Schimrigket al., Eur J Neurology 2006, 13, 604-610; and Schilling et al., ClinExperimental Immunology 2006, 145, 101-107); ischemia and reperfusioninjury (Joshi et al., US 2007/0027076); AGE-induced genome damage(Heidland, WO 2005/027899); inflammatory bowel diseases such as Crohn'sdisease and ulcerative colitis; arthritis; and others (Nilsson et al.,WO 2006/037342 and Nilsson and Muller, WO 2007/042034).

The mechanism of action of fumaric acid esters is believed to bemediated by pathways associated with the immunological response. Forexample, FAEs invoke a shift from a Th1 to Th2 immune response,favorably altering the cytokine profile; inhibit cytokine-inducedexpression of adhesion molecules such as VCAM-1, ICAM-1 and E-selectin,thereby reducing immune cell extravasation; and deplete lymphocytesthrough apoptotic mechanisms (Lehmann et al., J InvestigativeDermatology 2007, 127, 835-845; Gesser et al., J InvestigativeDermatology 2007, 127, 2129-2137; Vandermeeren et al., Biochm BiophysRes Commun 1997, 234, 19-23; and Treumer et al., J Invest Dermatol 2003,121, 1383-1388).

Recent studies suggest that FAEs are inhibitors of NF-κB activation, atranscription factor that regulates the inducible expression ofproinflammatory mediators (D'Acquisto et al., Molecular Interventions2002, 2(1), 22-35). Accordingly, FAEs have been proposed for use intreating NF-κB mediated diseases (Joshi et al., WO 2002/055066; andJoshi and Strebel, WO 2002/055063, US 2006/0205659, U.S. Pat. No.7,157,423 and U.S. Pat. No. 6,509,376). Inhibitors of NF-κB activationhave also been shown to be useful in angiostatic therapy (Tabruyn andGriffioen, Angiogenesis 2008, 11, 101-106), inflammatory bowel disease(Atreya et al., J Intern Med 2008, 263(6), 591-6); and in animal modelsof diseases involving inflammation including neutrophilic alveolitis,asthma, hepatitis, inflammatory bowel disease, neurodegeneration,ischemia/reperfusion, septic shock, glomerulonephritis, and rheumatoidarthritis (D'Acquisto et al., Molecular Interventions 2002, 2(1),22-35).

Studies also suggest that NF-κB inhibition by FAEs may be mediated byinteraction with tumor necrosis factor (TNF) signaling. Dimethylfumarate inhibits TNF-induced tissue factor mRNA and protein expressionand TNF-induced DNA binding of NF-κB proteins, and inhibits theTNF-induced nuclear entry of activated NF-κB proteins thereby inhibitinginflammatory gene activation (Loewe et al., J Immunology 2002, 168,4781-4787). TNF signaling pathways are implicated in the pathogenesis ofimmune-mediated inflammatory diseases such as rheumatoid arthritis,Crohn's disease, psoriasis, psoriatic arthritis, juvenile idiopathicarthritis, and ankylosing spondylitis (Tracey et al., Pharmacology &Therapeutics 2008, 117, 244-279).

Fumaderm®, an enteric coated tablet containing a salt mixture ofmonoethyl fumarate and dimethylfumarate (DMF) (2) which is rapidlyhydrolyzed to monomethyl fumarate (MHF) (1), regarded as the mainbioactive metabolite, was approved in Germany in 1994 for the treatmentof psoriasis.

Fumaderm® is dosed TID with 1-2 grams/day administered for the treatmentof psoriasis. Fumaderm® exhibits a high degree of interpatientvariability with respect to drug absorption and food strongly reducesbioavailability. Absorption is thought to occur in the small intestinewith peak levels achieved 5-6 hours after oral administration.Significant side effects occur in 70-90% of patients (Brewer and Rogers,Clin Expt'l Dermatology 2007, 32, 246-49; and Hoefnagel et al., Br JDermatology 2003, 149, 363-369). Side effects of current FAE therapyinclude gastrointestinal upset including nausea, vomiting, and diarrhea;transient flushing of the skin. Also, DMF exhibits poor aqueoussolubility.

Fumaric acid derivatives (Joshi and Strebel, WO 2002/055063, US2006/0205659, and U.S. Pat. No. 7,157,423 (amide compounds andprotein-fumarate conjugates); Joshi et al., WO 2002/055066 and Joshi andStrebel, U.S. Pat. No. 6,355,676 (mono and dialkyl esters); Joshi andStrebel, WO 2003/087174 (carbocyclic and oxacarbocylic compounds); Joshiet al., WO 2006/122652 (thiosuccinates); Joshi et al., US 2008/0233185(dialkyl and diaryl esters) and salts (Nilsson et al., US 2008/0004344)have been developed in an effort to overcome the deficiencies of currentFAE therapy. Controlled release pharmaceutical compositions comprisingfumaric acid esters are disclosed by Nilsson and Müller, WO 2007/042034.Glycolamide ester prodrugs are described by Nielsen and Bundgaard, JPharm Sci 1988, 77(4), 285-298.

SUMMARY

MHF prodrugs having high gastrointestinal permeability and/orabsorption, improved solubility, ordered hydrolysis (i.e., preferentialcleavage of promoieties), and minimal cleavage in the gut lumen orenterocyte cytoplasm are desirable. Such MHF prodrugs that providehigher oral bioavailability and plasma levels of MHF, DMF, and/or othermetabolites may enhance the efficacy/responder rate compared to presentfumaric acid esters; facilitate the use of lower doses, reduced dosingfrequency, and standardized dosing regimens; reduce food effects; reducegastrointestinal side effects/toxicity; and reduce interpatienttreatment variability.

In a first aspect, compounds of Formula (I) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl;

R³ and R⁴ are independently chosen from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl,C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂ cycloalkylalkyl, C₇₋₁₂arylalkyl, and substituted C₇₋₁₂ arylalkyl; or R³ and R⁴ together withthe nitrogen to which they are bonded form a ring chosen from a C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₅₋₁₀ heterocycloalkyl, andsubstituted C₅₋₁₀ heterocycloalkyl; and

R⁵ is chosen from methyl, ethyl, and C₃₋₆ alkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl;

with the proviso that when R⁵ is ethyl; then R³ and R⁴ are independentlychosen from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆ alkyl.

In a second aspect, compounds of Formula (II) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

R⁶ is chosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl,substituted C₁₋₆ heteroalkyl, C₃₋₈ cycloalkyl, substituted C₃₋₈cycloalkyl, C₆₋₈ aryl, substituted C₆₋₈ aryl, and —OR¹⁰ wherein R¹⁰ ischosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl,substituted C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and substituted C₆₋₁₀ aryl;

R⁷ and R⁸ are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl; and

R⁹ is chosen from C₁₋₆ alkyl and substituted C₁₋₆ alkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl.

In a third aspect, compounds of Formula (III) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl; and

R³ and R⁴ are independently chosen from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl,C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂ cycloalkylalkyl, C₇₋₁₂arylalkyl, and substituted C₇₋₁₂ arylalkyl; or R³ and R⁴ together withthe nitrogen to which they are bonded form a ring chosen from a C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₅₋₁₀ heterocycloalkyl, andsubstituted C₅₋₁₀ heterocycloalkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl.

In a fourth aspect, compounds of Formula (IV) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

R⁶ is chosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl,substituted C₁₋₆ heteroalkyl, C₃₋₈ cycloalkyl, substituted C₃₋₈cycloalkyl, C₆₋₈ aryl, substituted C₆₋₈ aryl, and —OR¹⁰, wherein R¹⁰ ischosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl,substituted C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and substituted C₆₋₁₀ aryl;and

R⁷ and R⁸ are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R^(a), —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl;

with the provisos that;

-   -   when one of R⁷ and R⁸ is chosen from ethyl and methyl, and the        other of R⁷ and R⁸ is hydrogen; then R⁶ is not —C(CH₃)═CH₂; and    -   when each of R⁷ and R⁹ is hydrogen; then R⁶ is not chosen from        —CH═CH₂ and 4-carboxyphenyl.

In a fifth aspect, pharmaceutical compositions are provided comprising acompound of Formulae (I)-(IV) and at least one pharmaceuticallyacceptable vehicle.

In a sixth aspect, methods of treating a disease in a patient areprovided comprising administering to a patient in need of such treatmenta pharmaceutical composition comprising a therapeutically effectiveamount of a compound of Formulae (I)-(IV). In certain embodiments, thedisease is chosen from psoriasis, multiple sclerosis, an inflammatorybowel disease, asthma, chronic obstructive pulmonary disease, andarthritis.

In a seventh aspect, methods of inhibiting NF-κB activation in a patientare provided comprising administering to a patient a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formulae (I)-(IV).

In an eighth aspect, methods of inhibiting TNF function in a patient areprovided comprising administering to a patient a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formulae (I)-(IV).

DETAILED DESCRIPTION Definitions

A dash (“—”) that is not between two letters or symbols is used toindicate a point of attachment for a moiety or substituent. For example,—CONH₂ is bonded through the carbon atom.

“Alkyl” refers to a saturated or unsaturated, branched, orstraight-chain, monovalent hydrocarbon radical derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkene,or alkyne. Examples of alkyl groups include, but are not limited to,methyl; ethyls such as ethanyl, ethenyl, and ethynyl; propyls such aspropan-1-yl, propan-2-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl(allyl), prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls suchas butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

The term “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds, and groupshaving combinations of single, double, and triple carbon-carbon bonds.Where a specific level of saturation is intended, the terms alkanyl,alkenyl, and alkynyl are used. In certain embodiments, an alkyl groupcan have from 1 to 20 carbon atoms (C₁₋₂₀) in certain embodiments, from1 to 10 carbon atoms (C₁₋₁₀), in certain embodiments from 1 to 8 carbonatoms (C₁₋₈), in certain embodiments, from 1 to 6 carbon atoms (C₁₋₆),in certain embodiments from 1 to 4 carbon atoms (C₁₋₄), and in certainembodiments, from 1 to 3 carbon atoms (C₁₋₃).

“Aryl” refers to a monovalent aromatic hydrocarbon radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Aryl benzene; bicyclic ring systems wherein atleast one ring is carbocyclic and aromatic, for example, naphthalene,indane, and tetralin; and tricyclic ring systems wherein at least onering is carbocyclic and aromatic, for example, fluorene. Arylencompasses multiple ring systems having at least one carbocyclicaromatic ring fused to at least one carbocyclic aromatic ring,cycloalkyl ring, or heterocycloalkyl ring. For example, aryl includes aphenyl ring fused to a 5- to 7-membered heterocycloalkyl ring containingone or more heteroatoms chosen from N, O, and S. For such fused,bicyclic ring systems wherein only one of the rings is a carbocyclicaromatic ring, the radical carbon atom may be at the carbocyclicaromatic ring or at the heterocycloalkyl ring. Examples of aryl groupsinclude, but are not limited to, groups derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like. In certain embodiments, an aryl group can have from 6 to20 carbon atoms (C₆₋₂₀), from 6 to 12 carbon atoms (C₆₋₁₂), from 6 to 10carbon atoms (C₆₋₁₀), and in certain embodiments from 6 to 8 carbonatoms (C₆₋₈). Aryl, however, does not encompass or overlap in any waywith heteroaryl, separately defined herein.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl group. Examples of arylalkylgroups include, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylalkenyl, or arylalkynyl is used. In certainembodiments, an arylalkyl group is C₇₋₃₀ arylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety of the arylalkyl group is C₁₋₁₀ and the arylmoiety is C₆₋₂₀, in certain embodiments, an arylalkyl group is C₆₋₁₈arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkylgroup is C₁₋₈ and the aryl moiety is C₆₋₁₀. In certain embodiments, anarylalkyl group is C₇₋₁₂ arylalkyl.

“Compounds” of Formulae (I)-(IV) disclosed herein include any specificcompounds within these formulae. Compounds may be identified either bytheir chemical structure and/or chemical name. Compounds are named usingChemistry 4-D Draw Pro, version 7.01c (ChemInnovation Software, Inc.,San Diego, Calif.). When the chemical structure and chemical nameconflict, the chemical structure is determinative of the identity of thecompound. The compounds described herein may comprise one or more chiralcenters and/or double bonds and therefore may exist as stereoisomerssuch as double-bond isomers (i.e., geometric isomers), enantiomers, ordiastereomers. Accordingly, any chemical structures within the scope ofthe specification depicted, in whole or in part, with a relativeconfiguration encompass all possible enantiomers and stereoisomers ofthe illustrated compounds including the stereoisomerically pure form(e.g., geometrically pure, enantiomerically pure, or diastereomericallypure) and enantiomeric and stereoisomeric mixtures. Enantiomeric andstereoisomeric mixtures may be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. Compounds of Formulae(I)-(IV) include, but are not limited to, optical isomers of compoundsof Formulae (I)-(IV), racemates thereof, and other mixtures thereof. Insuch embodiments, a single enantiomer or diastereomer, i.e., opticallyactive form can be obtained by asymmetric synthesis or by resolution ofthe racemates. Resolution of the racemates may be accomplished, forexample, by conventional methods such as crystallization in the presenceof a resolving agent, or chromatography using, for example, chiralstationary phases. Not withstanding the foregoing, in compounds ofFormulae (I)-(IV) the configuration of the illustrated double bond isonly in the E configuration (i.e. trans configuration).

Compounds of Formulae (I)-(IV) may also exist in several tautomericforms including the enol form, the keto form, and mixtures thereof.Accordingly, the chemical structures depicted herein encompass allpossible tautomeric forms of the illustrated compounds. Compounds ofFormulae (I)-(IV) also include isotopically labeled compounds where oneor more atoms have an atomic mass different from the atomic massconventionally found in nature. Examples of isotopes that may beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds mayexist in unsolvated forms as well as solvated forms, including hydratedforms and as N-oxides. In general, compounds as referred to herein maybe free acid, hydrated, solvated, or N-oxides. Certain compounds mayexist in multiple crystalline, co-crystalline, or amorphous forms.Compounds of Formulae (I)-(IV) include pharmaceutically acceptable saltsthereof, or pharmaceutically acceptable solvates of the free acid formof any of the foregoing, as well as crystalline forms of any of theforegoing.

Compounds of Formulae (I)-(IV) also include solvates. A solvate refersto a molecular complex of a compound with one or more solvent moleculesin a stoichiometric or non-stoichiometric amount. Such solvent moleculesare those commonly used in the pharmaceutical art, which are known to beinnocuous to a patient, e.g., water, ethanol, and the like. A molecularcomplex of a compound or moiety of a compound and a solvent can bestabilized by non-covalent intra-molecular forces such as, for example,electrostatic forces, van der Waals forces, or hydrogen bonds. The term“hydrate” refers to a solvate in which the one or more solvent moleculesis water.

Further, when partial structures of the compounds are illustrated, anasterisk (*) indicates the point of attachment of the partial structureto the rest of the molecule.

“Cycloalkyl” refers to a saturated or partially unsaturated cyclic alkylradical. Where a specific level of saturation is intended, thenomenclature cycloalkanyl or cycloalkenyl is used. Examples ofcycloalkyl groups include, but are not limited to, groups derived fromcyclopropane, cyclobutane, cyclopentane, cyclohexane, and the like. Incertain embodiments, a cycloalkyl group is C₃₋₁₅ cycloalkyl, C₃₋₁₂cycloalkyl, and in certain embodiments, C₃₋₈ cycloalkyl.

“Cycloalkylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a cycloalkyl group. Where specific alkylmoieties are intended, the nomenclature cycloalkylalkanyl,cycloalkylalkenyl, or cycloalkylalkynyl is used. In certain embodiments,a cycloalkylalkyl group is C₄₋₃₀ cycloalkylalkyl, e.g., the alkanyl,alkenyl, or alkynyl moiety of the cycloalkylalkyl group is C₁₋₁₀ and thecycloalkyl moiety is C₃₋₂₀, and in certain embodiments, acycloalkylalkyl group is C₃₋₂₀ cycloalkylalkyl, e.g., the alkanyl,alkenyl, or alkynyl moiety of the cycloalkylalkyl group is C₁₋₈ and thecycloalkyl moiety is C₃₋₁₂. In certain embodiments, a cycloalkylalkylgroup is C₄₋₁₂ cycloalkylalkyl.

“Disease” refers to a disease, disorder, condition, or symptom of any ofthe foregoing.

“Drug” as defined under 21 U.S.C. §321(g)(1) means “(A) articlesrecognized in the official United States Pharmacopoeia, officialHomeopathic Pharmacopoeia of the United States, or official NationalFormulary, or any supplement to any of them; and (B) articles intendedfor use in the diagnosis, cure, mitigation, treatment, or prevention ofdisease in man or other animals; and (C) articles (other than food)intended to affect the structure or any function of the body of man orother animals . . . .”

“Halogen” refers to a fluoro, chloro, bromo, or iodo group. In certainembodiments, halogen refers to a chloro group.

“Heteroalkyl” by itself or as part of another substituent refer to analkyl group in which one or more of the carbon atoms (and certainassociated hydrogen atoms) are independently replaced with the same ordifferent heteroatomic groups. Examples of heteroatomic groups include,but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR¹³, ═N—N═,—N═N—, —N═N—NR¹³—, —PR¹³—, —P(O)₂—, —POR¹³—, —O—P(O)₂—, —SO—, —SO₂—,—Sn(R¹³)₂—, and the like, where each R¹³ is independently chosen fromhydrogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₆₋₁₂ aryl, substitutedC₆₋₁₂ aryl, C₇₋₁₈ arylalkyl, substituted C₇₋₁₈ arylalkyl, C₃₋₇cycloalkyl, substituted C₃₋₇ cycloalkyl, C₃₋₇ heterocycloalkyl,substituted C₃₋₇ heterocycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₆₋₁₂ heteroaryl, substituted C₆₋₁₂ heteroaryl, C₇₋₁₈heteroarylalkyl, or substituted C₇₋₁₈ heteroarylalkyl. Reference to, forexample, a C₁₋₆ heteroalkyl, means a C₁₋₆ alkyl group in which at leastone of the carbon atoms (and certain associated hydrogen atoms) isreplaced with a heteroatom. For example C₁₋₆ heteroalkyl includes groupshaving five carbon atoms and one heteroatom, groups having four carbonatoms and two heteroatoms, etc. In certain embodiments, each R¹³ isindependently chosen from hydrogen and C₁₋₃ alkyl. In certainembodiments, a heteroatomic group is chosen from —O—, —S—, —NH—,—N(CH₃)—, and —SO₂—; and in certain embodiments, the heteroatomic groupis —O—.

“Heteroaryl” refers to a monovalent heteroaromatic radical derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Heteroaryl encompasses multiple ring systemshaving at least one heteroaromatic ring fused to at least one otherring, which can be aromatic or non-aromatic. For example, heteroarylencompasses bicyclic rings in which one ring is heteroaromatic and thesecond ring is a heterocycloalkyl ring. For such fused, bicyclicheteroaryl ring systems wherein only one of the rings contains one ormore heteroatoms, the radical carbon may be at the aromatic ring or atthe heterocycloalkyl ring. In certain embodiments, when the total numberof N, S, and O atoms in the heteroaryl group exceeds one, theheteroatoms are not adjacent to one another. In certain embodiments, thetotal number of heteroatoms in the heteroaryl group is not more thantwo.

Examples of heteroaryl groups include, but are not limited to, groupsderived from acridine, arsindole, carbazole, β-carboline, chromane,chromene, cinnoline, furan, imidazole, indazole, indole, indoline,indolizine, isobenzofuran, isochromene, isoindole, isoindoline,isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,oxazole, perimidine, phenanthridine, phenanthroline, phenazine,phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine,pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline,quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene,triazole, xanthene, thiazolidine, oxazolidine, and the like. In certainembodiments, a heteroaryl group is from 4- to 20-membered heteroaryl(C₄₋₂₀), and in certain embodiments from 4- to 12-membered heteroaryl(C₄₋₁₀). In certain embodiments, heteroaryl groups are those derivedfrom thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine,quinoline, imidazole, oxazole, or pyrazine. For example, in certainembodiments, C₅ heteroaryl can be furyl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, isothiazolyl, isoxazolyl.

“Heterocycloalkyl” refers to a saturated or unsaturated cyclic alkylradical in which one or more carbon atoms (and certain associatedhydrogen atoms) are independently replaced with the same or differentheteroatom; or to a parent aromatic ring system in which one or morecarbon atoms (and certain associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom such that the ring systemno longer contains at least one aromatic ring. Examples of heteroatomsto replace the carbon atom(s) include, but are not limited to, N, P, O,S, Si, etc. Examples of heterocycloalkyl groups include, but are notlimited to, groups derived from epoxides, azirines, thiuranes,imidazolidine, morpholine, piperazine, piperidine, pyrazolidine,pyrrolidine, quinuclidine, and the like. In certain embodiments, aheterocycloalkyl group is C₅₋₁₀ heterocycloalkyl, C₅₋₈ heterocycloalkyl,and in certain embodiments, C₅₋₆ heterocycloalkyl.

“Leaving group” has the meaning conventionally associated with it insynthetic organic chemistry, i.e., an atom or a group capable of beingdisplaced by a nucleophile and includes halogen such as chloro, bromo,fluoro, and iodo, acyloxy (alkoxycarbonyl) such as acetoxy andbenzoyloxy, aryloxycarbonyl, mesyloxy, tosyloxy,trifluoromethanesulfonyloxy, aryloxy such as 2,4-dinitrophenoxy,methoxy, N,O-dimethylhydroxylamino, p-nitrophenolate, imidazolyl, andthe like.

“Parent aromatic ring system” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π (pi) electron system.Included within the definition of “parent aromatic ring system” arefused ring systems in which one or more of the rings are aromatic andone or more of the rings are saturated or unsaturated, such as, forexample, fluorene, indane, indene, phenalene, etc. Examples of parentaromatic ring systems include, but are not limited to, aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Parent heteroaromatic ring system” refers to an aromatic ring system inwhich one or more carbon atoms (and any associated hydrogen atoms) areindependently replaced with the same or different heteroatom in such away as to maintain the continuous π-electron system characteristic ofaromatic systems and a number of out-of-plane π-electrons correspondingto the Hückel rule (4n+2). Examples of heteroatoms to replace the carbonatoms include, but are not limited to, N, P, O, S, and Si, etc.Specifically included within the definition of “parent heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, arsindole, benzodioxan, benzofuran, chromane,chromene, indole, indoline, xanthene, etc. Examples of parentheteroaromatic ring systems include, but are not limited to, arsindole,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,thiazolidine, oxazolidine, and the like.

“Patient” refers to a mammal, for example, a human.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the parent compound.Such salts include acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; andsalts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine, andthe like. In certain embodiments, a pharmaceutically acceptable salt isthe hydrochloride salt. In certain embodiments, a pharmaceuticallyacceptable salt is the sodium salt.

“Pharmaceutically acceptable vehicle” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable excipient, a pharmaceutically acceptablecarrier, or a combination of any of the foregoing with which a compoundprovided by the present disclosure may be administered to a patient andwhich does not destroy the pharmacological activity thereof and which isnon-toxic when administered in doses sufficient to provide atherapeutically effective amount of the compound.

“Pharmaceutical composition” refers to a compound of Formulae (I)-(IV)and at least one pharmaceutically acceptable vehicle, with which thecompound of Formulae (I)-(IV) is administered to a patient.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or substituent group(s). In certainembodiments, each substituent group is independently chosen fromhalogen, —OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NH₂, —R¹¹, —OR¹¹,—C(O)R¹¹, —COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosenfrom hydrogen and C₁₋₄ alkyl. In certain embodiments, each substituentgroup is independently chosen from halogen, —OH, —CN, —CF₃, —NO₂,benzyl, —R¹¹, —OR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independentlychosen from hydrogen and C₁₋₄ alkyl. In certain embodiments, eachsubstituent group is independently chosen from halogen, —OH, —CN, —CF₃,═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹, —COOR¹¹, and —NR¹¹₂ wherein each R¹¹ is independently chosen from hydrogen and C₁₋₄ alkyl.In certain embodiments, each substituent group is independently chosenfrom —OH, Clot alkyl, and —NH₂.

“Treating” or “treatment” of any disease refers to reversing,alleviating, arresting, or ameliorating a disease or at least one of theclinical symptoms of a disease, reducing the risk of acquiring a diseaseor at least one of the clinical symptoms of a disease, inhibiting theprogress of a disease or at least one of the clinical symptoms of thedisease or reducing the risk of developing a disease or at least one ofthe clinical symptoms of a disease. “Treating” or “treatment” alsorefers to inhibiting the disease, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both, and to inhibiting atleast one physical parameter that may or may not be discernible to thepatient. In certain embodiments, “treating” or “treatment” refers todelaying the onset of the disease or at least one or more symptomsthereof in a patient which may be exposed to or predisposed to a diseaseeven though that patient does not yet experience or display symptoms ofthe disease.

“Therapeutically effective amount” refers to the amount of a compoundthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease, is sufficient to affect suchtreatment of the disease or symptom thereof. The “therapeuticallyeffective amount” may vary depending, for example, on the compound, thedisease and/or symptoms of the disease, severity of the disease and/orsymptoms of the disease or disorder, the age, weight, and/or health ofthe patient to be treated, and the judgment of the prescribingphysician. An appropriate amount in any given instance may beascertained by those skilled in the art or capable of determination byroutine experimentation.

“Therapeutically effective dose” refers to a dose that provideseffective treatment of a disease or disorder in a patient. Atherapeutically effective dose may vary from compound to compound, andfrom patient to patient, and may depend upon factors such as thecondition of the patient and the route of delivery. A therapeuticallyeffective dose may be determined in accordance with routinepharmacological procedures known to those skilled in the art.

Reference is now made in detail to certain embodiments of compounds,compositions, and methods. The disclosed embodiments are not intended tobe limiting of the claims. To the contrary, the claims are intended tocover all alternatives, modifications, and equivalents.

Compounds

Certain embodiments provide a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl;

R³ and R⁴ are independently chosen from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl,C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂ cycloalkylalkyl, C₇₋₁₂arylalkyl, and substituted C₇₋₁₂ arylalkyl; or R³ and R⁴ together withthe nitrogen to which they are bonded form a ring chosen from a C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₅₋₁₀ heterocycloalkyl, andsubstituted C₅₋₁₀ heterocycloalkyl; and

R⁵ is chosen from methyl, ethyl, and C₃₋₆ alkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl;

with the proviso that when R⁵ is ethyl, then R³ and R⁴ are chosen fromhydrogen, C₁₋₆ alkyl, and substituted C₁₋₆ alkyl.

In certain embodiments of a compound of Formula (I), each substituentgroup is independently chosen from halogen, —OH, —CN, —CF₃, —R¹¹, —OR¹¹,and —NR¹¹ ₂ wherein each R¹¹ is independently chosen from hydrogen andC₁₋₄ alkyl. In certain embodiments, each substituent group isindependently chosen from —OH, and —COOH.

In certain embodiments of a compound of Formula (I), each substituentgroup is independently chosen from ═O, C₁₋₄ alkyl, and —COOR¹¹ whereinR¹¹ is chosen from hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (I), each of R¹ and R²is hydrogen.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is methyl.

In certain embodiments of a compound of Formula (I), R³ and R⁴ areindependently chosen from hydrogen and C₁₋₆ alkyl.

In certain embodiments of a compound of Formula (I), R³ and R⁴ areindependently chosen from hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (I), R³ and R⁴ areindependently chosen from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (I), each of R³ and R⁴is hydrogen; in certain embodiments, each of R³ and R⁴ is methyl; and incertain embodiments, each of R³ and R⁴ is ethyl.

In certain embodiments of a compound of Formula (I), R³ is hydrogen; andR⁴ is chosen from C₁₋₄ alkyl, substituted C₁₋₄ alkyl wherein thesubstituent group is chosen from ═O, —OR¹, —COOR¹¹, and —NR¹¹ ₂, whereineach R¹¹ is independently chosen form hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (I), R³ is hydrogen; andR⁴ is chosen from C₁₋₄ alkyl, benzyl, 2-methoxyethyl, carboxymethyl,carboxypropyl, 1,2,4-thiadoxolyl, methoxy, 2-methoxycarbonyl,2-oxo(1,3-oxazolidinyl), 2-(methylethoxy)ethyl, 2-ethoxyethyl,(tert-butyloxycarbonyl)methyl, (ethoxycarbonyl)methyl, carboxymethyl,(methylethyl)oxycarbonylmethyl, and ethoxycarbonylmethyl.

In certain embodiments of a compound of Formula (I), R³ and R⁴ togetherwith the nitrogen to which they are bonded form a ring chosen from aC₅₋₆ heterocycloalkyl, substituted C₅₋₆ heterocycloalkyl, C₅₋₆heteroaryl, and substituted C₅₋₆ heteroaryl ring. In certain embodimentsof a compound of Formula (I), R³ and R⁴ together with the nitrogen towhich they are bonded form a ring chosen from a C₅ heterocycloalkyl,substituted C₅ heterocycloalkyl, C₅ heteroaryl, and substituted C₅heteroaryl ring. In certain embodiments of a compound of Formula (I), R³and R⁴ together with the nitrogen to which they are bonded form a ringchosen from a C₆ heterocycloalkyl, substituted C₆ heterocycloalkyl, C₆heteroaryl, and substituted C₆ heteroaryl ring. In certain embodimentsof a compound of Formula (I), R³ and R⁴ together with the nitrogen towhich they are bonded form a ring chosen from piperazine,1,3-oxazolidinyl, pyrrolidine, and morpholine ring

In certain embodiments of a compound of Formula (I), R³ and R⁴ togetherwith the nitrogen to which they are bonded form a C₅₋₁₀ heterocycloalkylring.

In certain embodiments of a compound of Formula (I), R⁵ is methyl.

In certain embodiments of a compound of Formula (I), R⁵ is ethyl.

In certain embodiments of a compound of Formula (I), R⁵ is C₃₋₆ alkyl.

In certain embodiments of a compound of Formula (I), R⁵ is chosen frommethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, andtert-butyl.

In certain embodiments of a compound of Formula (I), R⁵ is chosen frommethyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, andtert-butyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is C₁₋₆ alkyl; R³ is hydrogen; R⁴ ischosen from hydrogen, C₁₋₆ alkyl, and benzyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is C₁₋₆ alkyl; R³ is hydrogen; R⁴ ischosen from hydrogen, C₁₋₆ alkyl, and benzyl; and R⁵ is methyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; and each of R³ and R⁴ is C₁₋₆ alkyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; each of R³ and R⁴ is C₁₋₆ alkyl; and R⁵ is methyl. In certainembodiments of a compound of Formula (I), each of R¹ and R² is hydrogen;each of R³ and R⁴ is C₁₋₆ alkyl; and R⁵ is methyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₄alkyl; R³ is hydrogen; R⁴ is chosen from C₁₋₄ alkyl, substituted C₁₋₄alkyl wherein the substituent group is chosen from ═O, —OR¹¹, —COOR¹¹,and —NR¹¹ ₂, wherein each R¹¹ is independently chosen form hydrogen andC₁₋₄ alkyl; and R⁵ is methyl. In certain embodiments of a compound ofFormula (I), one of R¹ and R² is hydrogen and the other of R¹ and R² ismethyl; R³ is hydrogen; R⁴ is chosen from C₁₋₄ alkyl, substituted C₁₋₄alkyl wherein the substituent group is chosen from ═O, —OR¹¹, —COOR¹¹,and —NR¹¹ ₂, wherein each R¹¹ is independently chosen form hydrogen andC₁₋₄ alkyl; and R⁵ is methyl. In certain embodiments of a compound ofFormula (I), each of R¹ and R² is hydrogen; R³ is hydrogen; R⁴ is chosenfrom C₁₋₄ alkyl, substituted C₁₋₄ alkyl wherein the substituent group ischosen from ═O, —OR¹¹, —COOR¹¹, and —NR¹¹ ₂, wherein each R¹¹ isindependently chosen form hydrogen and C₁₋₄ alkyl; and R⁵ is methyl.

In certain embodiments of a compound of Formula (I), R³ and R⁴ togetherwith the nitrogen to which they are bonded form a C₅₋₁₀ heterocycloalkylring.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; R³ and R⁴ together with the nitrogen to which they are bondedform a ring chosen from a C₅₋₆ heterocycloalkyl, substituted C₅₋₆heterocycloalkyl, C₅₋₆ heteroaryl, and substituted C₅₋₆ heteroaryl ring;and R⁵ is methyl. In certain embodiments of a compound of Formula (I),one of R¹ and R² is hydrogen and the other of R¹ and R² is methyl; R³and R⁴ together with the nitrogen to which they are bonded form a ringchosen from a C₅₋₆ heterocycloalkyl, substituted C₅₋₆ heterocycloalkyl,C₅₋₆ heteroaryl, and substituted C₅₋₆ heteroaryl ring; and R⁵ is methyl.In certain embodiments of a compound of Formula (I), each of R¹ and R²is hydrogen; R³ and R⁴ together with the nitrogen to which they arebonded form a ring chosen from a C₅₋₆ heterocycloalkyl, substituted C₅₋₆heterocycloalkyl, C₅₋₆ heteroaryl, and substituted C₅₋₆ heteroaryl ring;and R⁵ is methyl.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; and R³ and R⁴ together with the nitrogen to which they are bondedform a ring chosen from morpholine, piperazine, and N-substitutedpiperazine.

In certain embodiments of a compound of Formula (I), one of R¹ and R² ishydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; R³ and R⁴ together with the nitrogen to which they are bondedform a ring chosen from morpholine, piperazine, and N-substitutedpiperazine; and R⁵ is methyl.

In certain embodiments of a compound of Formula (I), R⁵ is not methyl.

In certain embodiments of a compound of Formula (I), R¹ is hydrogen, andin certain embodiments, R² is hydrogen.

In certain embodiments of a compound of Formula (I), the compound ischosen from:

-   (N,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   methyl[N-benzylcarbamoyl]methyl(2E)but-2-ene-1,4-dioate;-   methyl 2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate;-   (N-butylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   [N-(2-methoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate;-   2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}acetic    acid;-   4-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic    acid;-   methyl(N-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl(2E)but-2ene-1,4-dioate;-   (N,N-dimethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   (N-methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;    bis-(2-methoxyethylamino)carbamoyl]methyl    methyl(2E)but-2-ene-1,4-dioate;-   [N-(methoxycarbonyl)carbamoyl]methyl methyl(2E)but-2ene-1,4-dioate;-   4-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic    acid, sodium salt;-   methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate;-   methyl    2-oxo-2-(2-oxo(1,3-oxazolidin-3-yl)ethyl(2E)but-2ene-1,4-dioate;-   {N-[2-(dimethylamino)ethyl]carbamoyl}methyl methyl(2E)but-2ene-1,4    dioate;-   methyl 2-(4-methylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate;-   methyl    {N-[(propylamino)carbonyl]carbamoyl}methyl(2E)but-2ene-1,4-dioate;-   2-(4-acetylpiperazinyl)-2-oxoethyl methyl(2E)but-2ene-1,4-dioate;-   {N,N-bis[2-(methylethoxy)ethyl]carbamoyl}methyl    methyl(2E)but-2-ene-1,4-dioate;-   methyl 2-(4-benzylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate;-   [N,N-bis(2-ethoxyethyl)carbamoyl]methyl    methyl(2E)but-2-ene-1,4-dioate;-   2-{(2S)-2-[(tert-butyl)oxycarbonyl]pyrrolidinyl}-2-oxoethyl    methyl(2E)but-2ene-1,4-dioate;-   1-{2-{(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetyl}(2S)pyrrolidine-2-carboxylic    acid;-   (N-{[tert-butyl)oxycarbonyl]methyl}-N-methylcarbamoyl)methyl    methyl(2E)but-2ene1,4-dioate;-   {N-(ethoxycarbonyl)methyl]-N-methylcarbamoyl}methyl    methyl(2E)but-2-ene-1,4-dioate;-   methyl 1-methyl-2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate;-   [N,N-bis(2-methoxyethyl)carbamoyl]ethyl    methyl(2E)but-2-ene-1,4-dioate;-   (N,N-dimethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate;-   2-{2-[(2E)-3-(methoxy    carbonyl)prop-2-enoyloxyl]-N-methylacetylamino}acetic acid;-   (N-{[(tert-butyl)oxycarbonyl]methyl}carbamoyl)methyl    methyl(2E)but-2-ene-1,4-dioate;-   (2E)but-methyl-N-{[(methylethyl)oxycarbonyl]methyl}carbamoyl)methyl(2E)but-2-ene-1,4-dioate;-   {N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methyl    methyl(2E)but-2-ene-1,4-dioate;-   {N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}ethyl    methyl(2E)but-2-ene-1,4-dioate;-   {N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl}ethyl    methyl(2E)but-2-ene-1,4-dioate;-   (1S)-1-methyl-2-morpholin-4-yl-2-oxo ethyl    methyl(2E)but-2-ene-1,4-dioate;-   (1S)-1-[N,N-bis(2-methoxyethyl)carbamoyl]ethyl    methyl(2E)but-2-ene-1,4-dioate;-   (1R)-1-(N,N-diethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate;    and

a pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (I), the compound ischosen from:

-   (N,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   methyl[N-benzylcarbamoyl]methyl(2E)but-2-ene-1,4-dioate;-   methyl 2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate;-   (N-butylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   [N-(2-methoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate;-   2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}acetic    acid;-   {2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic    acid;-   methyl(N-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl(2E)but-2ene-1,4-dioate;-   (N,N-dimethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   (N-methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;-   bis-(2-methoxyethylamino)carbamoyl]methyl    methyl(2E)but-2-ene-1,4-dioate;-   [N-(methoxycarbonyl)carbamoyl]methyl methyl(2E)but-2ene-1,4-dioate;-   methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate;-   methyl    2-oxo-2-(2-oxo(1,3-oxazolidin-3-yl)ethyl(2E)but-2ene-1,4-dioate;-   {N-[2-(dimethylamino)ethyl]carbamoyl}methyl methyl(2E)but-2ene-1,4    dioate;-   (N-[(methoxycarbonyl)ethyl]carbamoyl)methyl    methyl(2E)but-2-ene-1,4-dioate;-   2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}propanoic    acid; and

a pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (I), R³ and R⁴ areindependently chosen from hydrogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₄₋₁₂ cycloalkylalkyl, substitutedC₄₋₁₂ cycloalkylalkyl, C₇₋₁₂ arylalkyl, substituted C₇₋₁₂ arylalkyl,C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₆₋₁₀ heteroaryl,substituted C₆₋₁₀ heteroaryl, C₄₋₁₂ heterocycloalkylalkyl, substitutedC₄₋₁₂ heterocycloalkylalkyl, C₇₋₁₂ heteroarylalkyl, substituted C₇₋₁₂heteroarylalkyl; or R³ and R⁴ together with the nitrogen to which theyare bonded form a ring chosen from a C₅₋₁₀ heteroaryl, substituted C₅₋₁₀heteroaryl, C₅₋₁₀ heterocycloalkyl, and substituted C₅₋₁₀heterocycloalkyl.

Certain embodiments provide a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R⁶ is chosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl,substituted C₁₋₆ heteroalkyl, C₃₋₈ cycloalkyl, substituted C₃₋₈cycloalkyl, C₆₋₈ aryl, substituted C₆₋₈ aryl, and —OR¹⁰ wherein R¹⁰ ischosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl,substituted C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and substituted C₆₋₁₀ aryl;

R⁷ and R⁸ are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl; and

R⁹ is chosen from C₁₋₆ alkyl and substituted C₁₋₆ alkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (II), each substituentgroup is independently chosen from halogen, —OH, —CN, —CF₃, —R¹¹, —OR¹¹,and —NR¹¹ ₂ wherein each R¹¹ is independently chosen from hydrogen andC₁₋₄ alkyl.

In certain embodiments of a compound of Formula (I), each substituentgroup is independently chosen from ═O, C₁₋₄ alkyl, and —COOR¹¹ whereinR¹¹ is chosen from hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (II), one of R⁷ and R⁸is hydrogen and the other of R⁷ and R⁸ is C₁₋₆ alkyl. In certainembodiments of a compound of Formula (II), one of R⁷ and R⁸ is hydrogenand the other of R⁷ and R⁸ is C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (II), one of R⁷ and R⁸is hydrogen and the other of R⁷ and R⁸ is chosen from methyl, ethyl,n-propyl, and isopropyl. In certain embodiments of a compound of Formula(II), each of R⁷ and R⁸ is hydrogen.

In certain embodiments of a compound of Formula (II), R⁹ is chosen fromsubstituted C₁₋₆ alkyl and —OR¹¹ wherein R¹¹ is independently C₁₋₄alkyl.

In certain embodiments of a compound of Formula (II), R⁹ is C₁₋₆ alkyl,in certain embodiments, R⁹ is C₁₋₃ alkyl; and in certain embodiments, R⁹is chosen from methyl and ethyl.

In certain embodiments of a compound of Formula (II), R⁹ is methyl.

In certain embodiments of a compound of Formula (II), R⁹ is chosen fromethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, andtert-butyl.

In certain embodiments of a compound of Formula (II), R⁹ is chosen frommethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

In certain embodiments of a compound of Formula (II), R⁶ is C₁₋₆ alkyl;one of R⁷ and R⁸ is hydrogen and the other of R⁷ and R⁸ is C₁₋₆ alkyl;and R⁹ is chosen from C₁₋₆ alkyl and substituted C₁₋₆ alkyl.

In certain embodiments of a compound of Formula (II), R⁶ is —OR¹⁰.

In certain embodiments of a compound of Formula (II), R¹⁰ is chosen fromC₁₋₄ alkyl, cyclohexyl, and phenyl.

In certain embodiments of a compound of Formula (II), R⁶ is chosen frommethyl, ethyl, n-propyl, and isopropyl; one of R⁷ and R⁸ is hydrogen andthe other of R⁷ and R⁸ is chosen from methyl, ethyl, n-propyl, andisopropyl.

In certain embodiments of a compound of Formula (II), R⁶ is substitutedC₁₋₂ alkyl, wherein each of the one or more substituent groups arechosen from —COOH, —NHC(O)CH₂NH₂, and —NH₂.

In certain embodiments of a compound of Formula (II), R⁶ is chosen fromethoxy, methylethoxy, isopropyl, phenyl, cyclohexyl, cyclohexylloxy,—CH(NH₂)CH₂COOH, CH₂CH(NH₂)COOH, —CH(NHC(O)CH₂NH₂)—CH₂COOH, and—CH₂CH(NHC(O)CH₂NH₂)—COOH.

In certain embodiments of a compound of Formula (II), R⁹ is chosen frommethyl and ethyl; one of R⁷ and R⁸ is hydrogen and the other of R⁷ andR⁸ is chosen from hydrogen, methyl, ethyl, n-propyl, and isopropyl; andR⁶ is chosen from C₁₋₃ alkyl, substituted C₁₋₂ alkyl wherein each of theone or more substituent groups are chosen —COOH, —NHC(O)CH₂NH₂, and—NH₂, —OR¹⁰ wherein R¹⁰ is chosen from C₁₋₃ alkyl and cyclohexyl,phenyl, and cyclohexyl.

In certain embodiments of a compound of Formula (II), the compound ischosen from:

-   ethoxycarbonyloxyethyl methyl(2E)but-2-ene-1,4-dioate;-   methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene-1,4-dioate;-   (cyclohexyloxycarbonyloxy)ethyl methyl(2E)but-2-ene-1,4-dioate; and

a pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (II), the compound ischosen from:

-   methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene-1,4-dioate;-   methyl phenylcarbonyloxyethyl(2E)but-2-ene-1,4-dioate;-   cyclohexylcarbonyloxybutyl methyl(2E)but-2-ene-1,4-dioate;-   [(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]ethyl    methyl(2E)but-2-ene-1,4-dioate;-   methyl 2-methyl-1-phenylcarbonyloxypropyl(2E)but-2-ene-1,4-dioate;    and

a pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (II), the compound ischosen from:

-   ethoxycarbonyloxyethyl methyl(2E)but-2-ene-1,4-dioate;-   methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene-1,4-dioate;-   methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene-1,4-dioate;-   methyl phenylcarbonyloxyethyl(2E)but-2-ene-1,4-dioate;-   cyclohexylcarbonyloxybutyl methyl(2E)but-2-ene-1,4-dioate;-   [(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]ethyl    methyl(2E)but-2-ene-1,4-dioate;-   (cyclohexyloxycarbonyloxy)ethyl methyl(2E)but-2-ene-1,4-dioate;-   methyl 2-methyl-1-phenylcarbonyloxypropyl(2E)but-2-ene-1,4-dioate;-   3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(3S)-3-aminopropanoic    acid, 2,2,2-trifluoroacetic acid;-   3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(2S)-2-aminopropanoic    acid, 2,2,2-trifluoroacetic acid;-   3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(3S)-3-(2-aminoacetylamino)propanoic    acid, 2,2,2-trifluoroacetic acid;-   3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(2S)-2-aminopropanoic    acid, 2,2,2-trifluoroacetic acid;-   3-{[(2E)-3-(methoxycarbonyl)prop-2enoyloxy]ethoxycarbonyloxy}(2S)-2-aminopropanoic    acid, chloride; and

a pharmaceutically acceptable salt of any of the foregoing.

Compounds provided by the present disclosure include compounds ofFormula (III) and Formula (IV). Compounds of Formula (III) and Formula(IV) may be produced by in vivo metabolism of compounds of Formula (I)and Formula (II), respectively; or may be administered to a patient.

Accordingly, certain embodiments provide a compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl; and

R³ and R⁴ are independently chosen from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl,C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂ cycloalkylalkyl, C₇₋₁₂arylalkyl, and substituted C₇₋₁₂ arylalkyl; or R³ and R⁴ together withthe nitrogen to which they are bonded form a ring chosen from a C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₅₋₁₀ heterocycloalkyl, andsubstituted C₅₋₁₀ heterocycloalkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), each substituentgroup is independently chosen from halogen, —OH, —CN, —CF₃, —R¹¹, —OR¹¹,and —NR¹¹ ₂ wherein each R¹¹ is independently chosen from hydrogen andC₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), each substituentgroup is independently chosen from ═O, C₁₋₄ alkyl, and —COOR¹¹ whereinR¹¹ is chosen from hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), each of R¹ and R²is hydrogen.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is chosen from methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is methyl.

In certain embodiments of a compound of Formula (III), R³ and R⁴ areindependently chosen from hydrogen and C₁₋₆ alkyl.

In certain embodiments of a compound of Formula (III), R³ and R⁴ areindependently chosen from hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), R³ and R⁴ areindependently chosen from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (III), each of R³ and R⁴is hydrogen; in certain embodiments, each of R³ and R⁴ is methyl; and incertain embodiments, each of R³ and R⁴ is ethyl.

In certain embodiments of a compound of Formula (III), R³ is hydrogen;and R⁴ is chosen from C₁₋₄ alkyl, substituted C₁₋₄ alkyl wherein thesubstituent group is chosen from ═O, —OR¹¹, —COOR¹¹, and —NR¹¹ ₂,wherein each R¹¹ is independently chosen form hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), R³ is hydrogen;and R⁴ is chosen from C₁₋₄ alkyl, benzyl, 2-methoxyethyl, carboxymethyl,carboxypropyl, 1,2,4-thiadoxolyl, methoxy, 2-methoxycarbonyl,2-oxo(1,3-oxazolidinyl), 2-(methylethoxy)ethyl, 2-ethoxyethyl,(tert-butyloxycarbonyl)methyl, (ethoxycarbonyl)methyl, carboxymethyl,(methylethyl)oxycarbonylmethyl, and ethoxycarbonylmethyl.

In certain embodiments of a compound of Formula (III), R³ and R⁴together with the nitrogen to which they are bonded form a ring chosenfrom a C₅₋₆ heterocycloalkyl, substituted C₅₋₆ heterocycloalkyl, C₅₋₆heteroaryl, and substituted C₅₋₆ heteroaryl ring. In certain embodimentsof a compound of Formula (III), R³ and R⁴ together with the nitrogen towhich they are bonded form a ring chosen from a C₅ heterocycloalkyl,substituted C₅ heterocycloalkyl, C₅ heteroaryl, and substituted C₅heteroaryl ring. In certain embodiments of a compound of Formula (III),R³ and R⁴ together with the nitrogen to which they are bonded form aring chosen from a C₆ heterocycloalkyl, substituted C₆ heterocycloalkyl,C₆ heteroaryl, and substituted C₆ heteroaryl ring. In certainembodiments of a compound of Formula (III), R³ and R⁴ together with thenitrogen to which they are bonded form a ring chosen from piperazine,1,3-oxazolidinyl, pyrrolidine, and morpholine ring

In certain embodiments of a compound of Formula (III), R³ and R⁴together with the nitrogen to which they are bonded form a C₅₋₁₀heterocycloalkyl ring.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is C₁₋₆ alkyl; R³ is hydrogen; R⁴is chosen from hydrogen, C₁₋₆ alkyl, and benzyl.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; and each of R³ and R⁴ is C₁₋₆ alkyl. In certain embodiments of acompound of Formula (III), each of R¹ and R² is hydrogen; and each of R³and R⁴ is C₁₋₆ alkyl. In certain embodiments of a compound of Formula(III), one of R¹ and R² is hydrogen and the other of R¹ and R² is chosenfrom hydrogen and C₁₋₄ alkyl; R³ is hydrogen; and R⁴ is chosen from C₁₋₄alkyl, substituted C₁₋₄ alkyl wherein the substituent group is chosenfrom ═O, —OR¹¹, —COOR¹¹, and —NR¹¹ ₂, wherein each R¹¹ is independentlychosen form hydrogen and C₁₋₄ alkyl. In certain embodiments of acompound of Formula (III), one of R¹ and R² is hydrogen and the other ofR¹ and R² is methyl; R³ is hydrogen; and R⁴ is chosen from C₁₋₄ alkyl,substituted C₁₋₄ alkyl wherein the substituent group is chosen from ═O,—OR¹¹, —COOR¹¹, and —NR¹¹ ₂, wherein each R¹ is independently chosenform hydrogen and C₁₋₄ alkyl. In certain embodiments of a compound ofFormula (III), each of R¹ and R² is hydrogen; R³ is hydrogen; and R⁴ ischosen from C₁₋₄ alkyl, substituted C₁₋₄ alkyl wherein the substituentgroup is chosen from ═O, —OR¹¹, —COOR¹¹, and —NR¹¹ ₂, wherein each R¹¹is independently chosen form hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (III), R³ and R⁴together with the nitrogen to which they are bonded form a C₅₋₁₀heterocycloalkyl ring.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; and R³ and R⁴ together with the nitrogen to which they are bondedform a ring chosen from a C₅₋₆ heterocycloalkyl, substituted C₅₋₆heterocycloalkyl, C₅₋₆ heteroaryl, and substituted C₅₋₆ heteroaryl ring.In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is methyl; and R³ and R⁴ togetherwith the nitrogen to which they are bonded form a ring chosen from aC₅₋₆ heterocycloalkyl, substituted C₅₋₆ heterocycloalkyl, C₅₋₆heteroaryl, and substituted C₅₋₆ heteroaryl ring. In certain embodimentsof a compound of Formula (III), each of R¹ and R² is hydrogen; and R³and R⁴ together with the nitrogen to which they are bonded form a ringchosen from a C₅₋₆ heterocycloalkyl, substituted C₅₋₆ heterocycloalkyl,C₅₋₆ heteroaryl, and substituted C₅₋₆ heteroaryl ring.

In certain embodiments of a compound of Formula (III), one of R¹ and R²is hydrogen and the other of R¹ and R² is chosen from hydrogen and C₁₋₆alkyl; and R³ and R⁴ together with the nitrogen to which they are bondedform a ring chosen from morpholine, piperazine, and N-substitutedpiperazine.

In certain embodiments of a compound of Formula (III), R³ and R⁴ areindependently chosen from hydrogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₄₋₁₂ cycloalkylalkyl, substitutedC₄₋₁₂ cycloalkylalkyl, C₇₋₁₂ arylalkyl, substituted C₇₋₁₂ arylalkyl,C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₆₋₁₀ heteroaryl,substituted C₆₋₁₀ heteroaryl, C₄₋₁₂ heterocycloalkylalkyl, substitutedC₄₋₁₂ heterocycloalkylalkyl, C₇₋₁₂ heteroarylalkyl, substituted C₇₋₁₂heteroarylalkyl; or R³ and R⁴ together with the nitrogen to which theyare bonded form a ring chosen from a C₅₋₁₀ heteroaryl, substituted C₅₋₁₀heteroaryl, C₅₋₁₀ heterocycloalkyl, and substituted C₅₋₁₀heterocycloalkyl.

In certain embodiments of a compound of Formula (III), R¹ is hydrogen,and in certain embodiments, R² is hydrogen.

In certain embodiments of a compound of Formula (III), the compound ischosen from:

-   (2E)-3-[(2-morpholin-4-yl-2-oxoethyl)oxycarbonyl]prop-2-enoic acid;-   (2E)-3-{[(N,N-diethylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic acid;-   (2E)-3-({[N-benzylcarbamoyl]methyl}oxycarbonyl) prop-2-enoic acid;-   (2E)-3-[(2-morpholin-4-yl-2-oxoethyl) oxycarbonyl]prop-2-enoic acid;-   (2E)-3-{[(N-butylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic acid;-   (2E-3-{[N-methoxy-N-methylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   bis-(2-methoxyethylamino)carbamoyl]methyl prop-2-enoic acid;-   N,N-dimethylcarbamoyl)methyl pro-2-enoic acid;-   (2E)-3-({[N-(3-carboxypropyl)carbamoyl]methyl}oxycarbonyl)    prop-2-enoic acid;-   (2E)but-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl prop-2-enoic acid;-   (2E)-3-[(2-{(2S)-2-[tert-butyl)    oxycarbonyl]pyrrolidinyl}-2-oxoethyl) oxycarbonyl]prop-2enoic acid;-   1-[2-((2E)-3-carboxyprop-2-enoyloxy)acetyl](2S)    pyrrolidine-2-carboxylic acid;-   (2E)-3-[([N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl}methyl)    oxycarbonyl]prop-2-enoic acid;-   (2E)-3-{[(N-{[(tert-butyl)    oxycarbonyl]methyl}-N-methylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-[(1-methyl-2-morpholin-4-yl-2-oxoethyl)    oxycarbonyl]prop-2-enoic acid;-   (2E)-3-({[N,N-bis(2-methoxyethyl)carbamoyl]ethyl}oxycarbonyl)    prop-2-enoic acid;-   (2E)-3-{[(N,N-dimethylcarbamoyl)ethyl]oxycarbonyl}prop-2-enoic acid;-   (2E)-3-[({N,N-bis[2-methylethoxy)ethyl]carbamoyl}methyl)    oxycarbonyl]prop-2-enoic acid;-   (2E)-3-({[N,N-bis(2-ethoxyethyl)carbamoyl]methyl}oxycarbonyl)    prop-2-enoic acid;-   (2E)-3-{[2-(4-acetylpiperazinyl)-2-oxoethyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-({2-oxo-2-[4-benzylpiperazinyl]ethyl}oxycarbonyl)prop-2-enoic    acid;-   (2E)-3-{[(N-{[(tert-butyl)oxycarbonyl]methyl}carbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-{[(N-methyl-N-{[(methylethyl)oxycarbonyl]methyl}carbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-[({N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methyl)oxycarbonyl]prop-2-enoic    acid;-   (2E)-3-[({N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}ethyl)oxycarbonyl]prop-2-enoic    acid;-   (2E)-3-[({N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl}ethyl)oxycarbonyl]prop-2-enoic    acid; and

a pharmaceutically acceptable salt of any of the foregoing.

Certain embodiments provide a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R⁶ is chosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl,substituted C₁₋₆ heteroalkyl, C₃₋₈ cycloalkyl, substituted C₃₋₈cycloalkyl, C₆₋₈ aryl, substituted C₆₋₈ aryl, and —OR¹⁰, wherein R¹⁰ ischosen from C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl,substituted C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, and substituted C₆₋₁₀ aryl;and

R⁷ and R⁸ are independently chosen from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl;

wherein each substituent group is independently chosen from halogen,—OH, —CN, —CF₃, ═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹,—COOR¹¹, and —NR¹¹ ₂ wherein each R¹¹ is independently chosen fromhydrogen and C₁₋₄ alkyl;

with the provisos that;

-   -   when one of R⁷ and R⁸ is chosen from ethyl and methyl, and the        other of R⁷ and R⁸ is hydrogen; then R⁶ is not —C(CH₃)═CH₂; and    -   when each of R⁷ and R⁸ is hydrogen; then R⁶ is not chosen from        —CH═CH₂ and 4-carboxyphenyl.

In certain embodiments of a compound of Formula (IV), each substituentgroup is independently chosen from halogen, —OH, —CN, —CF₃, —R¹¹, —OR¹¹,and —NR¹¹ ₂ wherein each R¹¹ is independently chosen from hydrogen andC₁₋₄ alkyl.

In certain embodiments of a compound of Formula (IV), each substituentgroup is independently chosen from ═O, C₁₋₄ alkyl, and —COOR¹¹ whereinR¹¹ is chosen from hydrogen and C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (IV), one of R⁷ and R⁸is hydrogen and the other of R⁷ and R⁸ is C₁₋₆ alkyl. In certainembodiments of a compound of Formula (IV), one of R⁷ and R⁸ is hydrogenand the other of R⁷ and R⁸ is C₁₋₄ alkyl.

In certain embodiments of a compound of Formula (IV), one of R⁷ and R⁸is hydrogen and the other of R⁷ and R⁸ is chosen from methyl, ethyl,n-propyl, and isopropyl. In certain embodiments of a compound of Formula(IV), each of R⁷ and R⁸ is hydrogen.

In certain embodiments of a compound of Formula (IV), R⁶ is C₁₋₆ alkyl;and one of R⁷ and R⁸ is hydrogen and the other of R⁷ and R⁸ is C₁₋₆alkyl.

In certain embodiments of a compound of Formula (IV), R⁶ is —OR¹⁰.

In certain embodiments of a compound of Formula (IV), R¹⁰ is chosen fromC₁₋₄ alkyl, cyclohexyl, and phenyl.

In certain embodiments of a compound of Formula (IV), R⁶ is chosen frommethyl, ethyl, n-propyl, and isopropyl; one of R⁷ and R⁸ is hydrogen andthe other of R⁷ and R⁸ is chosen from methyl, ethyl, n-propyl, andisopropyl.

In certain embodiments of a compound of Formula (IV), R⁶ is substitutedC₁₋₂ alkyl, wherein each of the one or more substituent groups arechosen from —COOH, —NHC(O)CH₂NH₂, and —NH₂.

In certain embodiments of a compound of Formula (IV), R⁶ is chosen fromethoxy, methylethoxy, isopropyl, phenyl, cyclohexyl, cyclohexyloxy,—CH(NH₂)CH₂COOH, —CH₂CH(NH₂)COOH, —CH(NHC(O)CH₂NH₂)—CH₂COOH, and—CH₂CH(NHC(O)CH₂NH₂)—COOH.

In certain embodiments of a compound of Formula (IV), one of R⁷ and R⁸is hydrogen and the other of R⁷ and R⁸ is chosen from hydrogen, methyl,ethyl, n-propyl, and isopropyl; and R⁶ is chosen from C₁₋₃ alkyl,substituted C₁₋₂ alkyl wherein each of the one or more substituentgroups are chosen COOH, —NHC(O)CH₂NH₂, and —NH₂, —OR¹⁰ wherein R¹⁰ ischosen from C₁₋₃ alkyl and cyclohexyl, phenyl, and cyclohexyl.

In certain embodiments of a compound of Formula (IV), the compound ischosen from:

-   (2E)-3-{[(2-methylpropanoyloxy)ethyl]oxycarbonyl}prop-2-enoic acid;-   (2E)-3-({[(methylethyl)oxycarbonyloxy]ethyl}oxycarbonyl)prop-2-enoic    acid;-   2-[(2E)-3-(methoxycarbonyl) prop-2-enoyloxy]acetic acid; and

a pharmaceutically acceptable salt of any of the foregoing.

Synthesis

Compounds disclosed herein may be obtained via the synthetic methodsillustrated in Schemes 1 through 9. General synthetic methods useful inthe synthesis of compounds described herein are available in the art.Starting materials useful for preparing compounds and intermediatesthereof and/or practicing methods described herein are commerciallyavailable or can be prepared by well-known synthetic methods. Themethods presented in the schemes provided by the present disclosure areillustrative rather than comprehensive. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of thedisclosure.

Certain of the unsubstituted, 1-mono-substituted or 1,1-bis-substitutedhalo acetamides useful for preparing compounds of Formula (I) areavailable from commercial sources. Non-commercially availableunsubstituted, 1-mono-substituted or 1,1-bis-substituted halo acetamidesuseful for preparing compounds of Formula (I) and intermediates thereofcan be prepared by well-known synthetic methods such as those describedin Schemes 1 and 2.

Functionalized 1-halo acetamides useful for the preparation of MHFacetamide prodrugs of Formula (I) can be prepared according to Scheme 1:

wherein X and Y are leaving groups such as halogen, and R¹, R², R³, andR⁴ are as defined herein. In certain embodiments of Scheme 1, X ischloro and Y is chloro or an O-acylisourea.

Chemical activation of the carboxylic acid to the correspondingcarboxylic acid chloride as shown in Scheme 1 can be achieved byreaction with chlorination agents such as thionyl chloride (SOCl₂),oxalyl chloride (C₂O₂Cl₂), or phosphorous pentachloride (PCl₅),optionally in the presence of a suitable catalyst such asN,N-dimethylformamide (DMF), and either in substance (absence ofsolvent) or in an inert organic solvent such as dichloromethane (DCM) atan appropriate temperature such as from about 0° C. to about 70° C.Chemical activation of the carboxylic acid can be performed in situ andwithout isolating the activated substrate prior to the followingaminolysis step. Optionally, the activated carboxylic acid can beisolated and/or purified using methods well known in the art, i.e.fractional distillation.

Alternatively, carbodiimide dehydration agents such asN,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC),or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, EDC),optionally in the presence of a catalytic or stoichiometric amount of asuitable additive such as 4-(N,N-dimethylaminopyridine (DMAP) (Steglichesterification conditions), 1-hydroxybenzotriazole (HOBt),1-hydroxy-7-aza-benzotriazole (HOAt), or N-hydroxysuccinimide (NHS);uronium or phosphonium salts with non-nucleophilic anions such asN-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmetanaminiumhexafluorophosphate (HBTU),N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmetanaminiumhexafluorophosphate N-oxide (HATU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmetanaminiumtetrafluoroborate (TBTU), orbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), can be employed to form an activated carboxylic acidderivative. Optionally, organic tertiary bases such as triethylamine(TEA) or diisopropylethylamine (DIEA) can also be employed. Theformation of the activated carboxylic acid derivative can take place inan inert solvent such as dichloromethane (DCM), N,N-dimethylformamide(DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA, DMAc), ormixtures of any of the foregoing at an appropriate temperature such asfrom about 0° C. to abut 40° C.

Aminolysis of in situ generated or isolated activated carboxylicderivatives with the appropriately functionalized amine derivative(HNR³R⁴) (Scheme 2) can take place in the presence of a suitable basesuch as an organic tertiary base, i.e., triethylamine (TEA),diethylaminoethylamine (DIEA), pyridine, or mixtures of any of theforegoing, optionally in the presence of suitable additives such asnucleophilic acylation catalysts, i.e., 4-(N,N-dimethylaminopyridine(DMAP), and in the same or other inert solvent as used for theactivation step such as dichloromethane (DCM), N,N-dimethylformamide(DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA, DMAc), ormixtures of any of the foregoing, at an appropriate temperature such asfrom about 0° C. to about 70° C.

Functionalized 1-hydroxy acetamides useful for the preparation of MHFacetamide prodrugs of Formula (I) can be also prepared according toScheme 2:

wherein PG is a hydroxyl protecting group; Y a leaving group such aschloro or an O-isourea derived radical; and R¹, R², R³, and R⁴ are asdefined herein.

Certain of the functionalized and activated 1-hydroxy acetic acidderivatives are commercially available, i.e., benzyloxyacetic acid andtert-butyloxy lactic acid. Methods for introducing hydroxyl protectinggroups (PGs) are well known in the art. Useful protecting groups totemporarily block the hydroxyl group of functionalized 1-hydroxy aceticacids include certain alkyl such as (substituted) benzyl ethers,tert-butyl ethers, trityl ether, or various silyl ethers such astert-butyl dimethylsilyl ether, triisopropylsilyl ether, ortert-butyldiphenylsilyl ethers.

Certain protected, functionalized and activated 1-hydroxy acetic acidderivatives are commercially available, i.e., benzyloxyacetyl chloride.Alternatively, the chemical activation of the protected andfunctionalized 1-hydroxy acetic acid derivative to the correspondingactivated carboxylic acid derivative, i.e., carboxylic acid chloride,O-acylisourea, activated esters, etc., can be achieved using similarreaction procedures and conditions as those described in Scheme 1 forthe activation of functionalized 1-halo acetic acid derivatives.

Aminolysis of in situ generated or isolated protected, functionalized,and activated 1-hydroxy acetic derivatives with functionalized amines(HNR³R⁴) can take place using similar reaction procedures and conditionsas those described in Scheme 1 for the aminolysis of functionalized,protected, and activated 1-halo acetic acid derivatives.

Orthogonal (or ordered) deprotection of the protected 1-hydroxyaceticacid derivative liberates the corresponding free hydroxyl group.Deprotection methods, procedures, and practices are well known in theart.

In certain embodiments, the protecting group can be an alkyl group suchas a tert-butyl group. Deprotection may be carried out by contacting atert-butyl protected functionalized 1-hydroxy acetamide derivative withan excess of a strong Brønsted acid such as trifluoroacetic acid (TFA)or hydrogen chloride (HCl) in an inert solvent such as dichloromethane(DCM), diethyl ether (Et₂O), 1,4-dioxane, or mixtures of any of theforegoing, at an appropriate temperature such as from about 0° C. toabout 40° C.

In certain embodiments, the protecting group can be selected from analkyl group such as a benzyl group. When the protecting group is abenzyl group, deprotection may be carried out by reacting thefunctionalized 1-hydroxy acetamide derivative with gaseous hydrogen (H₂)in the presence of a heterogenous catalyst, i.e., 5-10 wt-% palladium on(activated or wet coal), in a solvent such as methanol (MeOH), ethanol(EtOH), ethyl acetate (EtOAc), or mixtures of any of the foregoing,optionally in the presence of a small amount of an activator such as 1 Naq. hydrochloric acid at an appropriate temperature such as from about0° C. to about 40° C. and under a hydrogen atmosphere at a pressure ofabout 15 psi to about 60 psi.

Acetamide MHF prodrugs of Formula (I) can be prepared according toScheme 3:

wherein X is a leaving group such as halogen, and R¹, R², R³, R⁴, and R⁵are as defined herein. In certain embodiments of Scheme 3, X is chloroand R⁵ is alkyl such as methyl.

Nucleophilic displacement of the monoalkyl fumaric acid with thefunctionalized 1-halo acetamide (Scheme 1) as shown in Scheme 3 can takeplace in the presence of an inorganic base such as an alkali carbonatesuch as cesium hydrogencarbonate (CsHCO₃), cesium carbonate (Cs₂CO₃), orpotassium carbonate (K₂CO₃). Optionally, organic tertiary bases such astriethylamine (TEA), diisopropylethylamine (DIEA), or amidine;guanidine-based bases such as 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,1,3,3-tetramethylguanidine(TMG); silver salts such silver(I) oxide (Ag₂O) or silver(I) carbonate(Ag₂CO₃); or other halide scavengers known in the art can be employed.The corresponding alkali, tri- and tetraalkylammonoium, amidine, orguanide salts of the monoalkyl fumarate can be generated in situ or,alternatively, can be prepared separately. The reaction can take placein an inert solvent such as N,N-dimethylformamide (DMF),N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA, DMAc),dimethylsulfoxide (DMSO), tetrahydrofuran (THF), toluene, or mixtures ofany of the foregoing at an appropriate temperature such as from aboutroom temperature to about 70° C.

Acetamide MHF prodrugs of Formula (I) can also be prepared according toScheme 4:

wherein Y is a suitable leaving group such as halogen, an O-acylisourea,various triazolol esters, or others; and R¹, R², R³, R⁴, and R⁵ are asdefined herein. In certain embodiments of Scheme 4, Y is chloro and R⁵is alkyl such as methyl.

Chemical activation of the carboxylic acid to the correspondingcarboxylic acid chloride as shown in Scheme 4 can be accomplished byreaction with a chlorination agent such as thionyl chloride (SOCl₂),oxalyl chloride (C₂O₂Cl₂), phosphorous pentachloride (PCl₅), or others,optionally in the presence of a catalyst such as N,N-dimethylformamide(DMF), and either in substance (absence of solvent) or in an inertorganic solvent such as dichloromethane (DCM) at an appropriatetemperature such as from about 0° C. to about 70° C. Chemical activationof the carboxylic acid as shown in Scheme 4 can be performed in situwithout isolating the activated substrate prior to the subsequentalcoholysis step. Optionally, the activated carboxylic acid chloride canbe isolated and/or purified using methods well known in the art, i.e.fractional distillation.

Alternatively, carbodiimide dehydration agents such asN,N-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC),or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, EDC),optionally in the presence of a catalytic or stoichiometric amount of anadditive such as 4-(N,N-dimethylaminopyridine (DMAP) (Steglichesterification conditions), 1-hydroxybenzotriazole (HOBt),1-hydroxy-7-aza-benzotriazole (HOAt), or N-hydroxysuccinimide (HOSu); auronium or phosphonium salt with non-nucleophilic anions such asN-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmetanaminiumhexafluorophosphate (HBTU),N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmetanaminiumhexafluorophosphate N-oxide (HATU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmetanaminiumtetrafluoroborate (TBTU), orbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), can be employed to form an activated monoalkyl fumaratederivative. Optionally, organic tertiary bases such as triethylamine(TEA) or diethylaminoethylamine (DIEA) can also be employed. Theformation of activated monoalkyl fumarate derivatives can take place inan inert solvent such as dichloromethane (DCM), N,N-dimethylformamide(DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA, DMAc), ormixtures of any of the foregoing at an appropriate temperature such asfrom about room temperature to about 70° C.

Alcoholysis of the activated monoalkyl fumarate derivative with afunctionalized hydroxy acetamide derivative (Scheme 2) can take place inthe presence of a base, for example, an organic tertiary base such as,triethylamine (TEA), diethylaminoethylamine (DIEA), or pyridine,optionally in the presence of an additive such as a nucleophilicacylation catalyst, i.e., 4-(N,N-dimethylaminopyridine (DMAP) (Steglichesterification conditions), and in the same or other inert solvent asused for the activation step such as dichloromethane (DCM),N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP),N,N-dimethylacetamide (DMA, DMAc), or mixtures of any of the foregoingat an appropriate temperature such as from about 0° C. to about 70° C.

Acetamide MHF prodrugs of Formula (I) can also be prepared according toScheme 5:

wherein A is either a leaving group such as halogen or a nucleophiliccoupling group such as hydroxyl; Y is a leaving group such as halogen, aO-acylisourea, various triazolol esters, or others; PG is a carboxylprotecting group; and R¹, R², R³, R⁴, and R⁵ are as defined herein. Incertain embodiments of Scheme 5, X is bromo, PG is tert-butyl, each ofR¹ and R² is hydrogen, and the electrophile is tert-butyl bromoacetate.In certain embodiments of Scheme 5, Y is chloro or O-acylisourea derivedfrom 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), and R⁵ isalkyl such as methyl.

The nucleophilic displacement reaction of the monoalkyl fumaric acidwith a protected and functionalized 1-halo acetic acid derivative, i.e.,commercially available tert-butyl bromoacetate or others, can take placeusing similar reaction procedures and conditions as those described inScheme 3 for the direct formation of functionalized acetamide MHFprodrugs of Formula (I) from a monoalkyl fumaric acid and anappropriately functionalized 1-halo acetamide.

Alcoholysis of an intermediate activated monoalkyl fumaric acidderivative and a protected and functionalized 1-hydroxy acetic acidderivative can take place using similar reaction procedures andconditions as those used in Scheme 4 for the direct formation offunctionalized acetamide MHF prodrugs of Formula (I) from a monoalkylfumaric acid and an appropriately functionalized 1-hydroxy acetamide.

Orthogonal (or ordered) deprotection of a protected monoalkyl fumaricacid-functionalized acetic acid derivative liberates the correspondingfree monoalkyl fumarate ester intermediate bearing a free carboxylicacid moiety. When the protecting group is a tert-butyl group,deprotection may be carried out by contacting the tert-butyl protectedfumaric acid derivative with an excess of a strong Brønsted acid such astrifluoroacetic acid (TFA) or hydrogen chloride (HCl) in an inertsolvent such as dichloromethane (DCM), diethyl ether (Et₂O),1,4-dioxane, or mixtures of any of the foregoing, at an appropriatetemperature such as from about 0° C. to about 40° C.

Chemical activation of the liberated monoalkyl fumarate-functionalizedhydroxyacetic derivative (carboxylic acid) to the correspondingactivated carboxylic acid derivative, i.e., carboxylic acid chloride,O-acylisourea, activated esters, etc., can be accomplished usingreaction procedures and conditions similar to those described in Scheme4 for the activation of monoalkyl fumaric acid direct formation offunctionalized acetamide MHF prodrugs of Formula (I) from the monoalkylfumaric acid and the corresponding functionalized hydroxyl acetamide.

Aminolysis of in situ generated or isolated activated monoalkyl fumaratefunctionalized hydroxyacetic derivatives with functionalized amines(HNR³R⁴) can take place using reaction procedures and conditions similarto those described in Schemes 1 and 2 for the aminolysis of protected,suitably functionalized and activated hydroxy acetic acid derivatives.

Certain of the functionalized 1-haloalkyl carboxylates (1-acyloxyalkylhalides) or functionalized 1-alkoxycarbonyloxyalkyl halides useful forpreparing compounds of Formula (II) are available from commercialsources. Non-commercially available 1-haloalkyl carboxylates(1-acyloxyalkyl halides) or functionalized 1-alkoxycarbonyloxyalkylhalides can be prepared by methods well known in the art and are brieflydescribed in Schemes 6 and 7.

1-Acyloxyalky halides useful for the preparation of MHF prodrugs ofFormula (II) can be prepared according to Scheme 6:

wherein X is a leaving group such as halogen; and R⁶, R⁷, R⁸ are asdefined herein. In certain embodiments of Scheme 6, X is chloro and R⁶is 2-[methyl(2E)but-2-ene-4-ate]yl; one of R⁷ and R⁸ is hydrogen and theother of R⁷ and R⁸ is alkyl.

Functionalized 1-haloalkyl carboxylates (1-acyloxyalkyl halides) may beprepared by contacting a functionalized carboxylic acid halide such as acarboxylic acid chloride with a functionalized carbonyl compound such asan aldehyde in the presence of a Lewis acid catalyst such as anhydrouszinc chloride (ZnCl₂) in an inert solvent such as dichloromethane (DCM)at a temperature from about −10° C. to room temperature. The1-chloroalkyl carboxylates (1-acyloxyalkyl chlorides) may be useddirectly or may be isolated and purified by methods well known in theart such as by fractional distillation or silica gel columnchromatography.

1-Alkoxy- and 1-aryloxycarbonyloxyalkyl halides useful for thepreparation of MHF prodrugs of Formula (II) can be prepared according toScheme 7:

wherein X is a leaving group such as halogen, and R⁷, R⁸, and R¹⁰ are asdefined herein. In certain embodiments of Scheme 7, X is chloro and R¹⁰together with the oxygen atom to which it is bonded is equivalent to R⁶,which is defined herein.

Functionalized 1-alkoxy- or aryloxycarbonyloxyalkyl halides may beprepared by contacting a functionalized haloalkyl halo formate such as afunctionalized chloro alkyl- or aryl chloroformate with a functionalizedalcohol or phenol (HOR¹⁰) in the presence of a base such as an organicsecondary and tertiary base, i.e., dicyclohexyl amine (DCHA),triethylamine (TEA), diisopropylethylamine (DIEA, Hünigs-base),pyridine, in an inert solvent such as dichloromethane (DCM) at atemperature from about −10° C. to room temperature. The 1-alkoxy- oraryloxycarbonyloxyalkyl halides may be used directly or may be isolatedand purified by methods well known in the art such as by fractionaldistillation or silica gel column chromatography.

Acyloxyalkyl and alkoxycarbonyloxyalkyl MHF prodrugs of Formula (II) canbe prepared according to Scheme 8:

wherein X is a leaving group such as halogen, and R⁶, R⁷, R⁸, and R⁹ areas defined herein.

Nucleophilic displacement of the monoalkyl fumaric acid with afunctionalized 1-halo (Scheme 1) as shown in Scheme 8 can take place inthe presence of an inorganic base such as an alkali carbonate, i.e.,cesium bicarbonate (CsHCO₃), cesium carbonate (CS₂CO₃), or potassiumcarbonate (K₂CO₃). Alternatively, organic secondary and tertiary basessuch as dicyclohexyl amine (DCHA), triethylamine (TEA),diisopropylethylamine (DIEA), amidine or guanidine-based bases such as1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or1,1,3,3-tetramethylguanidine (TMG); silver salts such silver(I) oxide(Ag₂O) or silver(I) carbonate (Ag₂CO₃); or other halide scavengers knownin the art can be employed. The corresponding alkali, tri- andtetraalkylammonoium, amidine, or guanide salts of the monoalkyl fumaratecan be generated in situ or, alternatively, can be prepared separately.The reaction can take place in an inert solvent such asN,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP),N,N-dimethylacetamide (DMA, DMAc), dimethylsulfoxide (DMSO), ortetrahydrofuran (THF), toluene, or mixtures of any of the foregoing atan appropriate temperature such as from about room temperature to about70° C.

Fumaric acid glycolamide monoesters of Formula (III) or acyloxyalkyl-and alkoxy- or aryloxycarbonyloxyalkyl fumaric acid monoesters ofFormula (IV) can be prepared according to Scheme 9:

wherein X is a suitable leaving group such as chloro; and R¹, R², R³,R⁴, R⁶, R⁷, R⁸, and R⁹ are as defined herein. In certain embodiments ofScheme 9, X is chloro, and R⁹ is selected from hydrogen or tert-butyl(tBu) and the fumaric acid derivative is either fumaric acid or monotert-butyl fumarate.

The coupling of fumaric acid or monoalkyl fumarates, i.e., monotert-butyl fumarate, with functionalized 1-halo acetamide derivatives,functionalized 1-haloalkyl carboxylates (1-acyloxyalkyl halides), or1-alkoxy- or aryloxycarbonyloxyalkyl halides, can take place usingreaction procedures and conditions similar to those described in Schemes3 and 8 for the direct formation of functionalized acetamide MHFprodrugs of Formula (I) (Scheme 3) or acyloxyalkyl oralkoxy-aryloxycarbonyloxyalkyl MHF prodrugs of Formula (II) (Scheme 8).

In certain embodiments where R⁹ is alkyl, such as tert-butyl, orthogonal(or ordered) deprotection (or liberation of the free carboxylic acid)from the corresponding functionalized acetamide or acyloxyalkyl oralkoxy/aryloxycarbonyloxyalkyl tert-butyl fumarates may be accomplishedusing reaction procedures and conditions similar to those described inScheme 5.

Pharmaceutical Compositions

Pharmaceutical compositions provided by the present disclosure maycomprise a therapeutically effective amount of a compound of Formulae(I)-(IV) together with a suitable amount of one or more pharmaceuticallyacceptable vehicles so as to provide a composition for properadministration to a patient. Suitable pharmaceutical vehicles aredescribed in the art.

In certain embodiments, a compound of Formulae (I)-(IV) may beincorporated into pharmaceutical compositions to be administered orally.Oral administration of such pharmaceutical compositions may result inuptake of a compound of Formulae (I)-(IV) throughout the intestine andentry into the systemic circulation. Such oral compositions may beprepared in a manner known in the pharmaceutical art and comprise acompound of Formulae (I)-(IV) and at least one pharmaceuticallyacceptable vehicle. Oral pharmaceutical compositions may include atherapeutically effective amount of a compound of Formulae (I)-(IV) anda suitable amount of a pharmaceutically acceptable vehicle, so as toprovide an appropriate form for administration to a patient.

Compounds of Formulae (I)-(IV) may be incorporated into pharmaceuticalcompositions to be administered by any other appropriate route ofadministration including intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, oral, sublingual,intracerebral, intravaginal, transdermal, rectal, inhalation, ortopical.

Pharmaceutical compositions comprising a compound of Formulae (I)-(IV)and may be manufactured by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes. Pharmaceutical compositions maybe formulated in a conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients, or auxiliaries, whichfacilitate processing of compounds of Formulae (I)-(IV) or crystallineforms thereof and one or more pharmaceutically acceptable vehicles intoformulations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Pharmaceuticalcompositions provided by the present disclosure may take the form ofsolutions, suspensions, emulsion, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for administration to a patient.

Pharmaceutical compositions provided by the present disclosure may beformulated in a unit dosage form. A unit dosage form refers to aphysically discrete unit suitable as a unitary dose for patientsundergoing treatment, with each unit containing a predetermined quantityof a compound of Formulae (I)-(IV) calculated to produce an intendedtherapeutic effect. A unit dosage form may be for a single daily dose,for administration 2 times per day, or one of multiple daily doses,e.g., 3 or more times per day. When multiple daily doses are used, aunit dosage form may be the same or different for each dose. One or moredosage forms may comprise a dose, which may be administered to a patientat a single point in time or during a time interval.

Pharmaceutical compositions comprising a compound of Formulae (I)-(IV)may be formulated for immediate release.

In certain embodiments, an oral dosage form provided by the presentdisclosure may be a controlled release dosage form. Controlled deliverytechnologies can improve the absorption of a drug in a particular regionor regions of the gastrointestinal tract. Controlled drug deliverysystems may be designed to deliver a drug in such a way that the druglevel is maintained within a therapeutically effective window andeffective and safe blood levels are maintained for a period as long asthe system continues to deliver the drug with a particular releaseprofile in the gastrointestinal tract. Controlled drug delivery mayproduce substantially constant blood levels of a drug over a period oftime as compared to fluctuations observed with immediate release dosageforms. For some drugs, maintaining a constant blood and tissueconcentration throughout the course of therapy is the most desirablemode of treatment. Immediate release of drugs may cause blood levels topeak above the level required to elicit a desired response, which maywaste the drug and may cause or exacerbate toxic side effects.Controlled drug delivery can result in optimum therapy, and not only canreduce the frequency of dosing, but may also reduce the severity of sideeffects. Examples of controlled release dosage forms include dissolutioncontrolled systems, diffusion controlled systems, ion exchange resins,osmotically controlled systems, erodable matrix systems, pH independentformulations, gastric retention systems, and the like.

An appropriate oral dosage form for a particular pharmaceuticalcomposition provided by the present disclosure may depend, at least inpart, on the gastrointestinal absorption properties of a compound ofFormulae (I)-(IV) the stability of a compound of Formulae (I)-(IV) inthe gastrointestinal tract, the pharmacokinetics of a compound ofFormulae (I)-(IV) and the intended therapeutic profile. An appropriatecontrolled release oral dosage form may be selected for a particularcompound of Formulae (I)-(IV). For example, gastric retention oraldosage forms may be appropriate for compounds absorbed primarily fromthe upper gastrointestinal tract, and sustained release oral dosageforms may be appropriate for compounds absorbed primarily from the lowergastrointestinal tract. Certain compounds are absorbed primarily fromthe small intestine. In general, compounds traverse the length of thesmall intestine in about 3 to 5 hours. For compounds that are not easilyabsorbed by the small intestine or that do not dissolve readily, thewindow for active agent absorption in the small intestine may be tooshort to provide a desired therapeutic effect.

In certain embodiments, pharmaceutical compositions provided by thepresent disclosure may be practiced with dosage forms adapted to providesustained release of a compound of Formulae (I)-(IV) upon oraladministration. Sustained release oral dosage forms may be used torelease drugs over a prolonged time period and are useful when it isdesired that a drug or drug form be delivered to the lowergastrointestinal tract. Sustained release oral dosage forms include anyoral dosage form that maintains therapeutic concentrations of a drug ina biological fluid such as the plasma, blood, cerebrospinal fluid, or ina tissue or organ for a prolonged time period. Sustained release oraldosage forms include diffusion-controlled systems such as reservoirdevices and matrix devices, dissolution-controlled systems, osmoticsystems, and erosion-controlled systems. Sustained release oral dosageforms and methods of preparing the same are well known in the art.

An appropriate dose of a compound of Formulae (I)-(IV) or pharmaceuticalcomposition comprising a compound of Formulae (I)-(IV) may be determinedaccording to any one of several well-established protocols. For example,animal studies such as studies using mice, rats, dogs, and/or monkeysmay be used to determine an appropriate dose of a pharmaceuticalcompound. Results from animal studies may be extrapolated to determinedoses for use in other species, such as for example, humans.

Uses

Compounds of Formulae (I)-(IV) are prodrugs of MHF. Thus, compounds ofFormulae (I)-(IV) and pharmaceutical compositions thereof may beadministered to a patient suffering from any disease including adisorder, condition, or symptom for which MHF is known or hereafterdiscovered to be therapeutically effective. Indications for which MHFhas been prescribed, and hence for which a compound of Formulae(I)-(IV), or pharmaceutical compositions thereof are also expected to beeffective, include psoriasis. Other indications for which compounds ofFormulae (I)-(IV) may be therapeutically effective include multiplesclerosis, an inflammatory bowel disease, asthma, chronic obstructivepulmonary disease, and arthritis.

Methods of treating a disease in a patient provided by the presentdisclosure comprise administering to a patient in need of such treatmenta therapeutically effective amount of a MHF prodrug of Formulae(I)-(IV). Compounds of Formulae (I)-(IV) or pharmaceutical compositionsthereof may provide therapeutic or prophylactic plasma and/or bloodconcentrations of MHF following administration to a patient.

MHF prodrugs of Formulae (I)-(IV) may be included in a pharmaceuticalcomposition and/or dosage form adapted for oral administration, althoughMHF prodrug of Formulae (I)-(IV) may also be administered by any otherappropriate route, such as for example, by injection, infusion,inhalation, transdermal, or absorption through epithelial or mucosalmembranes (e.g., oral, rectal, and/or intestinal mucosa).

MHF prodrugs of Formulae (I)-(IV) may be administered in an amount andusing a dosing schedule as appropriate for treatment of a particulardisease. Daily doses of a MHF prodrug of Formulae (I)-(IV) may rangefrom about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about50 mg/kg, from about 1 mg/kg to about 50 mg/kg, and in certainembodiments, from about 5 mg/kg to about 25 mg/kg. In certainembodiments, MHF prodrugs of Formulae (I)-(IV) may be administered at adose over time from about 1 mg to about 5 g per day, from about 10 mg toabout 4 g per day, and in certain embodiments from about 20 mg to about2 g per day. An appropriate dose of a MHF prodrug of Formulae (I)-(IV)may be determined based on several factors, including, for example, thebody weight and/or condition of the patient being treated, the severityof the disease being treated, the incidence and/or severity of sideeffects, the manner of administration, and the judgment of theprescribing physician. Appropriate dose ranges may be determined bymethods known to those skilled in the art.

MHF prodrugs of Formulae (I)-(IV) may be assayed in vitro and in vivofor the desired therapeutic or prophylactic activity prior to use inhumans. In vivo assays, for example using appropriate animal models, mayalso be used to determine whether administration of a MHF prodrug ofFormulae (I)-(IV) is therapeutically effective.

In certain embodiments, a therapeutically effective dose of a MHFprodrug of Formulae (I)-(IV) may provide therapeutic benefit withoutcausing substantial toxicity including adverse side effects. Toxicity ofMHF prodrugs of Formulae (I)-(IV) and/or metabolites thereof may bedetermined using standard pharmaceutical procedures and may beascertained by those skilled in the art. The dose ratio between toxicand therapeutic effect is the therapeutic index. A dose of a MHF prodrugof Formulae (I)-(IV) may be within a range capable of establishing andmaintaining a therapeutically effective circulating plasma and/or bloodconcentration of a MHF prodrug of Formulae (I)-(IV) that exhibits littleor no toxicity.

MHF prodrug of Formulae (I)-(IV) may be used to treat diseases,disorders, conditions, and symptoms of any of the foregoing for whichMHF is known to provide or is later found to provide therapeuticbenefit. MHF is known to be effective in treating psoriasis, multiplesclerosis, an inflammatory bowel disease, asthma, chronic obstructivepulmonary disease, and arthritis. Hence, MHF prodrugs of Formulae(I)-(IV) may be used to treat any of the foregoing diseases anddisorders. The underlying etiology of any of the foregoing diseasesbeing treated may have a multiplicity of origins. Further, in certainembodiments, a therapeutically effective amount of one or more compoundsof Formulae (I)-(IV) may be administered to a patient, such as a human,as a preventative measure against various diseases or disorders. Thus, atherapeutically effective amount of one or more compounds of Formulae(I)-(IV) may be administered as a preventative measure to a patienthaving a predisposition for and/or history of immunological, autoimmune,and/or inflammatory diseases including psoriasis, asthma and chronicobstructive pulmonary diseases, cardiac insufficiency including leftventricular insufficiency, myocardial infarction and angina pectoris,mitochondrial and neurodegenerative diseases such as Parkinson'sdisease, Alzheimer's disease, Huntington's disease, retinopathiapigmentosa and mitochondrial encephalomyopathy, transplantationrejection, autoimmune diseases including multiple sclerosis, ischemiaand reperfusion injury, AGE-induced genome damage, inflammatory boweldiseases such as Crohn's disease and ulcerative colitis; and NF-κBmediated diseases.

Psoriasis

Psoriasis is characterized by hyperkeratosis and thickening of theepidermis as well as by increased vascularity and infiltration ofinflammatory cells in the dermis. Psoriasis vulgaris manifests assilvery, scaly, erythematous plaques on typically the scalp, elbows,knees, and buttocks. Guttate psoriasis occurs as tear-drop size lesions.

Fumaric acid esters are recognized for the treatment of psoriasis anddimethyl fumarate is approved for the systemic treatment of psoriasis inGermany (Mrowietz and Asadullah, Trends Mol Med 2005, 11(1), 43-48; andMrowietz et al., Br J Dermatology 1999, 141, 424-429).

Efficacy of MHF prodrugs for treating psoriasis can be determined usinganimal models and in clinical trials.

Inflammatory Arthritis

Inflammatory arthritis includes diseases such as rheumatoid arthritis,juvenile rheumatoid arthritis (juvenile idiopathic arthritis), psoriaticarthritis, and ankylosing spondylitis produce joint inflammation. Thepathogenesis of immune-mediated inflammatory diseases includinginflammatory arthritis is believed to involve TNF and NK-κB signalingpathways (Tracey et al., Pharmacology & Therapeutics 2008, 117,244-279). DMF has been shown to inhibit TNF and inflammatory diseasesincluding inflammatory arthritis are believed to involve TNF and NK-κBsignaling and therefore may be useful in treating inflammatory arthritis(Lowewe et al., J Immunology 2002, 168, 4781-4787).

The efficacy of MHF prodrugs for treating inflammatory arthritis can bedetermined using animal models and in clinical trials.

Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory autoimmune disease of thecentral nervous system caused by an autoimmune attack against theisolating axonal myelin sheets of the central nervous system.Demyelination leads to the breakdown of conduction and to severe diseasewith destruction of local axons and irreversible neuronal cell death.The symptoms of MS are highly varied with each individual patientexhibiting a particular pattern of motor, sensible, and sensorydisturbances. MS is typified pathologically by multiple inflammatoryfoci, plaques of demyelination, gliosis, and axonal pathology within thebrain and spinal cord, all of which contribute to the clinicalmanifestations of neurological disability (see e.g., Wingerchuk, LabInvest 2001, 81, 263-281; and Virley, NeuroRx 2005, 2(4), 638-649).Although the causal events that precipitate MS are not fully understood,evidence implicates an autoimmune etiology together with environmentalfactors, as well as specific genetic predispositions. Functionalimpairment, disability, and handicap are expressed as paralysis, sensoryand octintive disturbances spasticity, tremor, a lack of coordination,and visual impairment, which impact on the quality of life of theindividual. The clinical course of MS can vary from individual toindividual, but invariably the disease can be categorized in threeforms: relapsing-remitting, secondary progressive, and primaryprogressive.

Studies support the efficacy of FAEs for treating MS and are undergoingphase II clinical testing (Schimrigk et al., Eur J Neurology 2006, 13,604-610; and Wakkee and Thio, Current Opinion Investigational Drugs2007, 8(11), 955-962).

Assessment of MS treatment efficacy in clinical trials can beaccomplished using tools such as the Expanded Disability Status Scaleand the MS Functional as well as magnetic resonance imaging lesion load,biomarkers, and self-reported quality of life. Animal models of MS shownto be useful to identify and validate potential therapeutics includeexperimental autoimmune/allergic encephalomyelitis (EAE) rodent modelsthat simulate the clinical and pathological manifestations of MS andnonhuman primate EAE models.

Inflammatory Bowel Disease (Crohn's Disease, Ulcerative Colitis)

Inflammatory bowel disease (IBD) is a group of inflammatory conditionsof the large intestine and in some cases, the small intestine thatincludes Crohn's disease and ulcerative colitis. Crohn's disease, whichis characterized by areas of inflammation with areas of normal lining inbetween, can affect any part of the gastrointestinal tract from themouth to the anus. The main gastrointestinal symptoms are abdominalpain, diarrhea, constipation, vomiting, weight loss, and/or weight gain.Crohn's disease can also cause skin rashes, arthritis, and inflammationof the eye. Ulcerative colitis is characterized by ulcers or open soresin the large intestine or colon. The main symptom of ulcerative colitisis typically constant diarrhea with mixed blood of gradual onset. Othertypes of intestinal bowel disease include collagenous colitis,lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet'scolitis, and indeterminate colitis.

FAEs are inhibitors of NF-κB activation and therefore may be useful intreating inflammatory diseases such as Crohn's disease and ulcerativecolitis (Atreya et al., J Intern Med 2008, 263(6), 59106).

The efficacy of MHF prodrugs for treating inflammatory bowel disease canbe evaluated using animal models and in clinical trials. Useful animalmodels of inflammatory bowel disease are known.

Asthma

Asthma is reversible airway obstruction in which the airway occasionallyconstricts, becomes inflamed, and is lined with an excessive amount ofmucus. Symptoms of asthma include dyspnea, wheezing, chest tightness,and cough. Asthma episodes may be induced by airborne allergens, foodallergies, medications, inhaled irritants, physical exercise,respiratory infection, psychological stress, hormonal changes, coldweather, or other factors.

As an inhibitor of NF-κB activation and as shown in animal studies(Joshi et al., US 2007/0027076) FAEs may be useful in treating pulmonarydiseases such as asthma and chronic obstructive pulmonary disorder.

The efficacy of MHF prodrugs of Formulae (I)-(IV) for treating asthmacan be assessed using animal models and in clinical trials.

Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD), also known as chronicobstructive airway disease, is a group of diseases characterized by thepathological limitation of airflow in the airway that is not fullyreversible, and includes conditions such as chronic bronchitis,emphysema, as well as other lung disorders such as asbestosis,pneumoconiosis, and pulmonary neoplasms (see, e.g., Barnes,Pharmacological Reviews 2004, 56(4), 515-548). The airflow limitation isusually progressive and associated with an abnormal inflammatoryresponse of the lungs to noxious particles and gases. COPD ischaracterized by a shortness of breath the last for months or years,possibly accompanied by wheezing, and a persistent cough with sputumproduction. COPD is most often caused by tobacco smoking, although itcan also be caused by other airborne irritants such as coal dust,asbestos, urban pollution, or solvents. COPD encompasses chronicobstructive bronchiolitis with fibrosis and obstruction of smallairways, and emphysema with enlargement of airspaces and destruction oflung parenchyma, loss of lung elasticity, and closure of small airways.

The efficacy of administering at least one compound of Formula (I) orFormula (II) for treating chronic obstructive pulmonary disease may beassessed using animal models of chronic obstructive pulmonary diseaseand in clinical studies. For example, murine models of chronicobstructive pulmonary disease are known.

Neurodegenerative Disorders

Neurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease, Huntington's disease and amyoptrophic lateral sclerosis arecharacterized by progressive dysfunction and neuronal death. NF-κBinhibition has been proposed as a therapeutic target forneurodegenerative diseases (Camandola and Mattson, Expert Opin TherTargets 2007, 11(2), 123-32).

Parkinson's Disease

Parkinson's disease is a slowly progressive degenerative disorder of thenervous system characterized by tremor when muscles are at rest (restingtremor), slowness of voluntary movements, and increased muscle tone(rigidity). In Parkinson's disease, nerve cells in the basal ganglia,e.g., substantia nigra, degenerate, and thereby reduce the production ofdopamine and the number of connections between nerve cells in the basalganglia. As a result, the basal ganglia are unable to smooth musclemovements and coordinate changes in posture as normal, leading totremor, incoordination, and slowed, reduced movement (bradykinesia)(Blandini, et al., Mol. Neurobiol. 1996, 12, 73-94).

The efficacy of compounds of Formulae (I)-(IV) for treating Parkinson'sdisease may be assessed using animal and human models of Parkinson'sdisease and in clinical studies.

Alzheimer's Disease

Alzheimer's disease is a progressive loss of mental functioncharacterized by degeneration of brain tissue, including loss of nervecells and the development of senile plaques and neurofibrillary tangles.In Alzheimer's disease, parts of the brain degenerate, destroying nervecells and reducing the responsiveness of the maintaining neurons toneurotransmitters. Abnormalities in brain tissue consist of senile orneuritic plaques, e.g., clumps of dead nerve cells containing anabnormal, insoluble protein called amyloid, and neurofibrillary tangles,twisted strands of insoluble proteins in the nerve cell.

The efficacy of compounds of Formulae (I)-(IV) for treating Alzheimer'sdisease may be assessed using animal and human models of Alzheimer'sdisease and in clinical studies.

Huntington's Disease

Huntington's disease is an autosomal dominant neurodegenerative disorderin which specific cell death occurs in the neostriatum and cortex(Martin, N Engl J Med 1999, 340, 1970-80). Onset usually occurs duringthe fourth or fifth decade of life, with a mean survival at age of onsetof 14 to 20 years. Huntington's disease is universally fatal, and thereis no effective treatment. Symptoms include a characteristic movementdisorder (Huntington's chorea), cognitive dysfunction, and psychiatricsymptoms. The disease is caused by a mutation encoding an abnormalexpansion of CAG-encoded polyglutamine repeats in the protein,huntingtin.

The efficacy of compounds of Formulae (I)-(IV) for treating Huntington'sdisease may be assessed using animal and human models of Huntington'sdisease and in clinical studies.

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerativedisorder characterized by the progressive and specific loss of motorneurons in the brain, brain stem, and spinal cord (Rowland andSchneider, N Engl J Med 2001, 344, 1688-1700). ALS begins with weakness,often in the hands and less frequently in the feet that generallyprogresses up an arm or leg. Over time, weakness increases andspasticity develops characterized by muscle twitching and tightening,followed by muscle spasms and possibly tremors. The average age of onsetis 55 years, and the average life expectancy after the clinical onset is4 years. The only recognized treatment for ALS is riluzole, which canextend survival by only about three months.

The efficacy compounds of Formulae (I)-(IV) for treating ALS may beassessed using animal and human models of ALS and in clinical studies.

Others

Other diseases and conditions for which compounds of Formulae (I)-(IV)can be useful in treating include rheumatica, granuloma annulare, lupus,autoimmune carditis, eczema, sarcoidosis, and autoimmune diseasesincluding acute disseminated encephalomyelitis, Addison's disease,alopecia greata, ankylosing spondylitis, antiphospholipid antibodysyndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneinner ear disease, bullous pemphigoid, Behcet's disease, celiac disease,Chagas disease, chronic obstructive pulmonary disease, Crohn's disease,dermatomyositis, diabetes mellitus type I, endometriosis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease,hidradenitis suppurativea, Kawasaki disease, IgA neuropathy, idiopathicthrombocytopenic purpura, interstitial cystitis, lupus erythematosus,mixed connective tissue disease, morphea, multiple sclerosis, myastheniagravis, narcolepsy, neuromyotonia, pemphigus vulgaris, perniciousanaemia, psoriasis, psonatic arthritis, polymyositis, primary biliarycirrhosis, rheumatoid arthritis, schizophrena, scleroderma, Sjogren'ssyndrome, stiff person syndrome, temporal arteritis, ulcerative colitis,vasculitis, vitiligo, and Wegener's granulomatosis.

Administration

MHF prodrugs of Formulae (I)-(IV) and pharmaceutical compositionsthereof may be administered orally or by any other appropriate route,for example, by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal, andintestinal mucosa, etc.). Other suitable routes of administrationinclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intracerebral, intravaginal, transdermal, rectal,inhalation, or topical.

Administration may be systemic or local. Various delivery systems areknown, e.g., encapsulation in liposomes, microparticles, microcapsules,capsules, etc.) that may be used to administer a compound and/orpharmaceutical composition.

The amount of a MHF prodrug of Formulae (I)-(IV) that will be effectivein the treatment of a disease in a patient will depend, in part, on thenature of the condition and can be determined by standard clinicaltechniques known in the art. In addition, in vitro or in vivo assays maybe employed to help identify optimal dosage ranges. A therapeuticallyeffective amount of a MHF prodrug of Formulae (I)-(IV) to beadministered may also depend on, among other factors, the subject beingtreated, the weight of the subject, the severity of the disease, themanner of administration, and the judgment of the prescribing physician.

For systemic administration, a therapeutically effective dose may beestimated initially from in vitro assays. For example, a dose may beformulated in animal models to achieve a beneficial circulatingcomposition concentration range. Initial doses may also be estimatedfrom in vivo data, e.g., animal models, using techniques that are knownin the art. Such information may be used to more accurately determineuseful doses in humans. One having ordinary skill in the art mayoptimize administration to humans based on animal data.

A dose may be administered in a single dosage form or in multiple dosageforms. When multiple dosage forms are used the amount of compoundcontained within each dosage form may be the same or different. Theamount of a MHF prodrug of Formulae (I)-(IV) contained in a dose maydepend on the route of administration and whether the disease in apatient is effectively treated by acute, chronic, or a combination ofacute and chronic administration.

In certain embodiments an administered dose is less than a toxic dose.Toxicity of the compositions described herein may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. In certain embodiments, a MHF prodrug may exhibit a hightherapeutic index. The data obtained from these cell culture assays andanimal studies may be used in formulating a dosage range that is nottoxic for use in humans. A dose of a MHF prodrug provided by the presentdisclosure may be within a range of circulating concentrations in forexample the blood, plasma, or central nervous system, that include theeffective dose and that exhibits little or no toxicity. A dose may varywithin this range depending upon the dosage form employed and the routeof administration utilized. In certain embodiments, an escalating dosemay be administered.

Combination Therapy

Methods provided by the present disclosure further compriseadministering one or more pharmaceutically active compounds in additionto a MHF prodrug of Formulae (I)-(IV). Such compounds may be provided totreat the same disease or a different disease than the disease beingtreated with the MHF prodrug of Formulae (I)-(IV).

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) may be usedin combination with at least one other therapeutic agent. In certainembodiments, a MHF prodrug of Formulae (I)-(IV) may be administered to apatient together with another compound for treating diseases andconditions involving immunological, autoimmune, and/or inflammatoryprocesses including: psoriasis; asthma, chronic obstructive pulmonarydiseases, and arthritis; cardiac insufficiency including leftventricular insufficiency, myocardial infarction and angina pectoris;mitochondrial and neurodegenerative diseases such as Parkinson'sdisease, Alzheimer's disease, Huntington's disease, retinopathiapigmentosa and mitochondrial encephalomyopathy; transplantationrejection; autoimmune diseases including multiple sclerosis (MS);ischemia and reperfusion injury (AGE-induced genome damage; and others.In certain embodiments, a MHF prodrug of Formulae (I)-(IV) may beadministered to a patient together with another compound for treatingpsoriasis, multiple sclerosis, an inflammatory bowel disease, asthma,chronic obstructive pulmonary disease, and arthritis.

A MHF prodrug of Formulae (I)-(IV) and the at least one othertherapeutic agent may act additively or, and in certain embodiments,synergistically. The at least one additional therapeutic agent may beincluded in the same dosage form as a MHF prodrug of Formulae (I)-(IV)or may be provided in a separate dosage form. Methods provided by thepresent disclosure can further include, in addition to administering aMHF prodrug of Formulae (I)-(IV), administering one or more therapeuticagents effective for treating the same or different disease than thedisease being treated by a MHF prodrug of Formulae (I)-(IV). Methodsprovided by the present disclosure include administration of a MHFprodrug of Formulae (I)-(IV) and one or more other therapeutic agentsprovided that the combined administration does not inhibit thetherapeutic efficacy of the MHF prodrug and/or does not typicallyproduce significant and/or substantial adverse combination effects.

In certain embodiments, dosage forms comprising a MHF prodrug ofFormulae (I)-(IV) may be administered concurrently with theadministration of another therapeutic agent, which may be part of thesame dosage form as, or in a different dosage form than that comprisinga MHF prodrug of Formulae (I)-(IV). A MHF prodrug of Formulae (I)-(IV)may be administered prior or subsequent to administration of anothertherapeutic agent. In certain embodiments of combination therapy, thecombination therapy may comprise alternating between administering a MHFprodrug of Formulae (I)-(IV) and a composition comprising anothertherapeutic agent, e.g., to minimize adverse drug effects associatedwith a particular drug. When a MHF prodrug of Formulae (I)-(IV) isadministered concurrently with another therapeutic agent thatpotentially may produce an adverse drug effect including, but notlimited to, toxicity, the other therapeutic agent may advantageously beadministered at a dose that falls below the threshold at which theadverse drug reaction is elicited.

In certain embodiments, dosage forms comprising a MHF prodrug ofFormulae (I)-(IV) may be administered with one or more substances toenhance, modulate and/or control release, bioavailability, therapeuticefficacy, therapeutic potency, stability, and the like of a MBF prodrugof Formulae (I)-(IV). For example, to enhance the therapeutic efficacyof a MHF prodrug ligand of Formulae (I)-(IV), the MHF prodrug ofFormulae (I)-(IV) may be co-administered with or a dosage formcomprising a MHF prodrug of Formulae (I)-(IV) may comprise one or moreactive agents to increase the absorption or diffusion of a MHF prodrugof Formulae (I)-(IV) from the gastrointestinal tract to the systemiccirculation, or to inhibit degradation of the MHF prodrug of Formulae(I)-(IV) in the blood of a patient. In certain embodiments, a MHFprodrug of Formulae (I)-(IV) may be co-administered with an active agenthaving pharmacological effects that enhance the therapeutic efficacy ofa MHF prodrug of Formulae (I)-(IV).

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient fortreating psoriasis in combination with a therapy or another therapeuticagent known or believed to be effective in treating psoriasis. Drugsuseful for treating psoriasis include steroids such as flurandrenolide,fluocinonide, alclometasone, amcinonide, desonide, halcinonide,triamcinolone, clobetasol, clocortolone, mometasone, desoximetasone, andhalobetasol; anti-rheumatics such as etanercept, infiximab, andadalimumab; immunosuppressive agents such as cyclosporine, alefacept,and efalizumab; psoralens such as methoxsalen; and other such ascalcipotriene, methotrexate, hydrocortisone/pramoxine, acitretin,betamethasone/calcipotriene, tazaraotene, benzocaine/pyrilamine/zincoxide, and ustekinumab.

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient fortreating inflammatory arthritis such as rheumatoid arthritis, juvenilerheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis incombination with a therapy or another therapeutic agent known orbelieved to be effective in treating inflammatory arthritis such asrheumatoid arthritis, juvenile rheumatoid arthritis, psoriaticarthritis, and ankylosing spondylitis.

Drugs useful for treating rheumatoid arthritis include non-steroidalanti-inflammatory agents such as ibuprofen, ketoprofen, salicylate,diclofenac, nabumetone, naproxen, meloxicam, sulindac, flurbiprofen,indomethacin, tolmetin, piroxicam, fenoprofen, oxaprozin, and etodolac;antiheumatics such as entanercept, adalimumab, infliximab,hydroxychloroquine, leflunomide, azathioprine, penicillamine,methotrexate, anakinra, auranofin, rituximab, aurothioglucose,tocilizumab, and golimumab; cox-2 inhibtors such as celecoxib andvadecoxib; corticosteroids such as triamcinolone; glucocorticoids suchas methylprednisolone and prednisone; and others such as sulfasalazine.

Drugs useful for treating juvenile rheumatoid arthritis includeadalimumab, abatacept, and infliximab.

Drugs useful for treating psoriatic arthritis include etanercept,adalimumab, triamcinolone, cortisone, infliximab, and golimumab.

Drugs useful for treating ankylosing spondylitis include adalimumab,celecoxib, diclofenac, etanercept, golimumab, indomethacin infliximab,naptoxen, olsalazine, salicylates, sulfindac, and triamcinolone.

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient fortreating psoriatic arthritis in combination with a therapy or anothertherapeutic agent known or believed to be effective in treatingpsioriatic arthritis. Drugs useful for treating psioriatic arthritisinclude etanercept, adalimumab, triamcinolone, cortisone, infliximab,and golimumab.

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient fortreating autoimmune diseases such as lupus in combination with a therapyor another therapeutic agent known or believed to be effective intreating autoimmune diseases such as lupus. Drugs useful for treatinglupus include hydroxychlooquine, triamcinolone, salicylate,azathioprine, and abetimus.

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient fortreating multiple sclerosis in combination with a therapy or anothertherapeutic agent known or believed to be effective in treating multiplesclerosis. Drugs useful for treating multiple sclerosis includeinterferon β-1a, interferon β-1b, glatiramer, modafinil, azathioprine,predisolone, mycophenolate mofetil, mitoxantrone, and natalizumab. Otherexamples of drugs useful for treating MS include Examples of drugsuseful for treating MS include corticosteroids such asmethylprednisolone; IFN-β such as IFN-β1a and IFN-β1b; glatirameracetate; monoclonal antibodies that bind to the very late antigen-4(VLA-4) integrin such as natalizumab; immunomodulatory agents such asFTY 720 sphinogosie-1 phosphate modulator and COX-2 inhibitors such asBW755c, piroxicam, and phenidone; and neuroprotective treatmentsincluding inhibitors of glutamate excitotoxicity and iNOS, free-radicalscavengers, and cationic channel blockers; memantine; AMPA antagonistssuch as topiramate; and glycine-site NMDA antagonists.

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient fortreating inflammatory bowel disease in combination with a therapy oranother therapeutic agent known or believed to be effective in treatinginflammatory bowel disease. Drugs useful for treating inflammatory boweldisease include cromolyn and mercaptopurine; and more particularly fortreating Crohn's disease include certolizumab, budesonide, azathioprine,sulfasalazine, metronidazole, adalimumab, mercaptopurine, infliximab,mesalamine, and natalizumab; and for treating ulcerative colitis includebalsalazide, infliximab, azathioprine, mesalamine, and cyclosporine.

In certain embodiments, MHF prodrugs provided by the present disclosureand pharmaceutical compositions thereof may be administered to a patientfor treating asthma in combination with a therapy or another therapeuticagent known or believed to be effective in treating asthma, or incertain embodiments, a disease, disorder, or condition associated withasthma. Examples of drugs useful in treating asthma include albuterol,aminophylline, beclomethasone, bitolterol, budesonide, cromolyn,ephedrine, epinephrine, flunisolide, fluticasone, formoterol,hydrocortisone, isoproterenol, levalbuterol, methylprednisolone,prednisolone, prednisone, pirbuterol, metaproterenol, racepinephrine,omalizumab, oxytriphylline, mometusone, montelukast, nedocromil,oxtriphylline, pirbuterol, salmeterol, terbutaline, theophylline,triamcinolone, zafirlukast, and zileuton.

In certain embodiments, MHF prodrugs provided by the present disclosureand pharmaceutical compositions thereof may be administered to a patientfor treating chronic obstructive pulmonary disease in combination with atherapy or another therapeutic agent known or believed to be effectivein treating chronic obstructive pulmonary disease, or in certainembodiments, a disease, disorder, or condition associated with chronicobstructive pulmonary disease. Examples of drugs useful for treatingchronic obstructive pulmonary disease include albuterol, arformoterol,azithromycin, bitolterol, epinephrine, fluticasone, formoterol,ipratropium, isoproterenol, levabuterol, metaproterenol, pirbuterol,racepinephrine, salmeterol, and tiotropium. Useful drugs for treatingchronic obstructive pulmonary disease further include bronchodialatorssuch as β2 agonists such as salbutamol, bambuterol, clenbuterol,fenoterol, and formoterol; M3 antimuscarinics such as ipratropium;leukotriene antagonists such as montelukast, pranlukast, andzafirlukast; cromones such as cromoglicate and nedocromil; xanthinessuch as theophylline; corticosteroids such as beclomethasone,mometasone, and fluticasone; and TNF antagonists such as infliximab,adalimumab, and etanercept. Other treatments for chronic obstructivepulmonary disease include oxygen therapy, and pulmonary rehabilitation.

In certain embodiments, prodrugs provided by the present disclosure andpharmaceutical compositions thereof may be administered to a patient fortreating angiogenesis in combination with a therapy or anothertherapeutic agent known or believed to be effective in treatingangiogenesis. Useful drugs for treating angiogenesis includeangiostatin, endostatin, vitaxin, bevacizumab, thalidomide, batimastat,marimastat, carboxyamidotraizole, TNP-470, CM101, IFN-α, IL-12, plateletfactor-4, suramin, SU5416, thrombospondin, VEGFR, angiostatic steroids,cartilage-derived angiogenesis inhibitory factor, matrixmetalloproteinase inhibitors, 2-methoxyestradiol, tecogalan,thrombospondin, prolactin, α_(v)β₃ inhibitors, and linomide.

In certain embodiments, prodrugs provided by the present disclosure andpharmaceutical compositions thereof may be administered to a patient fortreating transplant rejection in combination with a therapy or anothertherapeutic agent known or believed to be effective in treatingtransplant rejection. Useful drugs for treating transplant rejectioninclude calcineurin inhibitors such as cyclosporine and tacrolimus, mTORinhibitors such as sirolimus and everolimus, anti-proliferatives such asazathioprine and mycophenolic acid; corticosteroids such as monoclonalanti-IL2Rα receptor antibodies including basiliximab and daclizumab; andpolyclonal anti-T-cell antibodies including anti-thymocyte globulin andanti-lymphocyte globulin.

In certain embodiments, prodrugs provided by the present disclosure andpharmaceutical compositions thereof may be administered to a patient fortreating transplantation rejection in combination with a therapy oranother therapeutic agent known or believed to be effective in treatingtransplantation rejection. Examples of drugs useful in transplantationrejection include corticosteroids such as dexamethasone, prednisolone,and prednisone; globulins such as antilymphocyte globulin andantithymocyte globulin; macrolide immunosuppressants such as sirolimus,tacrolimus, and everolimus; mitotic inhibitors such as azathiprine,cylophosphamide, and methotrexate; monoclonal antibodies such asbasiliximab, daclizumab, infliximab, muromonoab; fungal metabolites suchas cyclosporine; and others such as glatiramer and mycophenolate.

In certain embodiments, prodrugs provided by the present disclosure andpharmaceutical compositions thereof may be administered to a patient fortreating cardiac insufficiency in combination with a therapy or anothertherapeutic agent known or believed to be effective in treating cardiacinsufficiency. Useful drugs for treating cardiac insufficiency includeantitensin-modulating agents, diuretics such as furosemide, bumetanie,hydrochlorothiazide, chlorthalidone, chlorthiazide, spironolactone,eplerenone: beta blockers such as bisoprolol, carvedilol, andmetroprolol; positive inotropes such as digoxin, milrinone, anddobutamine; alternative vasodilators such as isosorbidedinitrate/hydralazine; aldosterone receptor antagonists; recombinantneuroendocrine hormones such as nesiritide; and vasopressin receptorantagonists such as tolvaptan and conivaptan.

In certain embodiments, prodrugs provided by the present disclosure andpharmaceutical compositions thereof may be administered to a patient fortreating a mitochondrial disease such as a neurodegenerative disorder incombination with a therapy or another therapeutic agent known orbelieved to be effective in treating a mitochondrial disease such as aneurodegenerative disorder. In certain embodiments, a neurodegenerativedisorder is chosen from Alzheimer's disease, Parkinson's disease,Huntington's disease, and amyotrophic lateral sclerosis.

Therapeutic agents useful for treating Parkinson's disease includedopamine precursors such levodopa, dopamine agonists such asbromocriptine, pergolide, pramipexole, and ropinirole, MAO-B inhibitorssuch as selegiline, anticholinergic drugs such as benztropine,trihexyphenidyl, tricyclic antidepressants such as amitriptyline,amoxapine, clomipramine, desipramine, doxepin, imipramine, maprotiline,nortriptyline, protriptyline, amantadine, and trimipramine, someantihistamines such as diphenhydramine; antiviral drugs such asamantadine; and beta blockers such as propranolol.

Useful drugs for treating Alzheimer's disease include rosiglitazone,roloxifene, vitamin E, donepezil, tacrine, rivastigmine, galantamine,and memantine.

Useful drugs for treating symptoms of Huntington's disease includeantipsychotics such as haloperidol, chiorpromazine and olanzapine tocontrol hallucinations, delusions and violent outbursts; antidepressantssuch as fluoxetine, sertraline, and nortryiptyline to control depressionand obsessive-compulsive behavior; tranquilizers such asbenzodiazepines, paroxetine, venflaxin and beta-blockers to controlanxiety and chorea; mood stabilizers such as lithium, valproate, andcarbamzepine to control mania and bipolar disorder; and botulinum toxinto control dystonia and jaw clenching. Useful drugs for treatingsymptoms of Huntington's disease further include selective serotoninreuptake inhibitors (SSRI) such as fluoxetine, paroxetine, sertraline,escitalopram, citalopram, fluvosamine; norepinephrine and serotoiunreuptake inhibitors (NSRI) such as venlafaxine and duloxetine,benzodiazepines such as clonazepam, alprazolam, diazepam, and lorazepam,tricyclic antidepressants such as amitriptyline, nortnriptyline, andimipramine; and atypical antidepressants such as busipirone, bupriopion,and mirtazepine for treating the symptoms of anxiety and depression;atomoxetine, dextroamphetamine, and modafinil for treating apathysymptoms; amantadine, memantine, and tetrabenazine for treating choreasymptoms; citalopram, atomoxetine, memantine, rivastigmine, anddonepezil for treating cognitive symptoms; lorazepam and trazedone fortreating insomma; valproate, carbamazepine and lamotrigine for treatingsymptoms of irritability; SSRI antidepressants such as fluoxetine,paroxetine, sertaline, and fluvoxamine, NSRI antidepressants such asvenlafaxine, and others such as mirtazepine, clomipramine, lomotrigine,gabapentin, valproate, carbamazepine, olanzapine, rispiridone, andquetiapine for treating symptoms of obsessive-compulsive disorder;haloperidol, quetiapine, clozapine, risperidone, olanzapine,ziprasidone, and aripiprazole for treating psychosis; and pramipexole,levodopa and amantadine for treating rigidity.

Useful drugs for treating ALS include riluzole. Other drugs of potentialuse in treating ALS include memantine, tamoxifen, thalidomide,ceftriaxone, sodium phenyl butyrate, celecoxib, glatiramer acetate,busipirone, creatine, minocycline, coenzyme Q10, oxandrolone, IGF-1,topiramate, xaliproden, and indinavir. Drugs such as baclofen anddiazepam can be useful in treating spasticity associated with ALS.

In certain embodiments, a MHF prodrug of Formulae (I)-(IV) or apharmaceutical composition thereof may be administered to a patient incombination with a therapy or another therapeutic agent known orbelieved to be effective in inhibiting TNF function.

Examples of drugs known to inhibit TNF function include infliximab,adalimumab, etanercept, certolizumab, goliimumab, pentoxifylline,quanylhydrozone, thalidomide, flavonoids such as narigenin, resveratoland quecetin, alkaloids such as lycorine, terpenes such as acanthoicacid, fatty acids such as 13-HOA, and retinoids such as retinoic acid.

EXAMPLES

The following examples describe in detail the synthesis of MHF prodrugsof Formulae (I)-(IV), properties of MHF prodrugs of Formulae (I)-(IV),and uses of MHF prodrugs of Formulae (I)-(IV). It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of thedisclosure.

General Experimental Protocols

All reagents and solvents that were purchased from commercial supplierswere used without further purification or manipulation procedures.

Proton NMR (400 MHz) and carbon NMR spectra (125 MHz) were recorded on aVarian AS 400 NMR spectrometer equipped with an autosampler and dataprocessing software. CDCl₃ (99.8% D), DMSO-d⁶ (99.9% D), or MeOH-d⁴(99.8+% D), and acetonitrile-d³ were used as solvents unless otherwisenoted. The CHCl₃, DMSO-d⁵, or MeOH-d³ solvent signals were used forcalibration of the individual spectra. Analytical thin layerchromatography (TLC) was performed using a Whatman, Schleicher & SchuellT LC and MK6F silica gel plates (2.5×7.5 cm, 250 μm layer thickness).Melting points were recorded in glass capillaries using a StanfordResearch Systems (SRS) Optimelt Automated Melting Point System, S/N78047. Analytical LC/MS was performed on a Waters 2790 separation moduleequipped with a Waters Micromass QZ mass spectrometer, a Waters 996photodiode detector, and a Merck Chromolith UM2072-027 or PhenomenexLuna C-18 analytical column. Mass-guided preparative HPLC purificationof final compounds was performed using an instrument equipped with aWaters 600 controller, ZMD Micromass spectrometer, a Waters 2996photodiode array detector, and a Waters 2700 Sample Manager.Acetonitrile/water gradients containing 0.05% formic acid were used aseluents in both analytical and preparative HPLC experiments. Compoundisolation from aqueous solvent mixtures, e.g., acetonitrile/water/0.05%formic acid, was accomplished by primary lyophilization (freeze drying)of the frozen solutions under reduced pressure at room temperature usingmanifold freeze dryers such as a Heto Drywinner DW 6-85-1, a Heto FD4,or a VIRTIS Freezemobile 25 ES equipped with high vacuum pumps. When theisolated compound had ionizable functional groups such as an amino groupor a carboxylic acid, lyophilization was performed in the presence of aslight excess of one molar (1 M) hydrochloric acid to yield the purifiedcompounds as the corresponding hydrochloride salts (HCl-salts) or thecorresponding protonated free carboxylic acids. When the isolatedcompound had ionizable functional groups such as a carboxylic acid,lyophilization was performed in the presence of equimolar amounts ofsodium hydrogen carbonate (NaHCO₃) to yield the purified compounds asthe corresponding sodium salts (Na-salts). Optionally, the isolatedmaterials were further purified by flush silica gel columnchromatography, optionally employing Biotage pre-packed silica gelcartridges. Suitable organic solvents such as ethyl acetate (EtOAc),hexane (Hxn), n-heptane (Hptn), or mixtures and/or gradients thereofwere used as eluents to yield the target compounds as colorless, viscousoils or solids after evaporation of the solvents. Chemical names weregenerated with the Chemistry 4-D Draw Pro Version 7.01c (Draw ChemicalStructures Intelligently© 1993-2002) from ChemInnovation Software, Inc.,San Diego, USA).

Non-commercially available appropriately functionalized or substituted2-haloacetamides, 2-halo acetic acid derivatives, 2-hydroxy acetamides,2-hydroxy acetic acid derivatives, acyloxyalkyl halides, or alkoxy- oraryloxycarbonyloxyalkyl halides were synthesized from commerciallyavailable starting materials, and by adapting methods well known in theart.

General Synthetic Procedures

General Procedure A: Nucleophilic Substitution of 1-haloacetamides or1-halo Acetic Acid Derivatives with Monomethyl Fumarate:

(2E)-3-(Methoxycarbonyl)prop-2-enoic acid (methyl hydrogen fumarate,MHF), (2E)-3-(tert-butoxycarbonyl)prop-2-enoic acid (tert-butyl hydrogenfumarate), or fumaric acid (FA) (1.0 equivalents) is dissolved in 5-10mL/3.0 mmol of an inert solvent such as N-methylpyrrolidone (NMP),N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA, DMAc),acetonitrile (MeCN), dimethylsulfoxide (DMSO), tetrahydrofuran (THF),toluene, or mixtures thereof. To the solution, 0.8 to 1.2 equivalents ofan appropriate inorganic base such as cesium hydrogen carbonate(CsHCO₃), cesium carbonate (Cs₂CO₃), or potassium carbonate (K₂CO₃) isadded. Alternatively, 0.8 b is 1.2 equivalents of a silver salt suchsilver(I) oxide (Ag₂O) or silver(I) carbonate (Ag₂CO₃); an organicsecondary or tertiary base such as dicyclohexylamine (DCHA),triethylamine (TEA), diisopropylethylamine (DIEA), tetrabutylammoniumhydroxide (TBAOH), amidine; or a guanidine-based base such as1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or1,1,3,3-tetramethylguanidine (TMG), can be employed. The correspondingalkali, silver, di-, tri- and tetraalkylammonium, amidine, or guanidesalt of monoalkyl fumarate can also be preformed. The solution isstirred for 10-60 min at room temperature followed by addition of0.8-1.2 equivalents of an appropriately functionalized 1-haloacetamide,1-halo acetic acid derivative, acyloxyalkyl halide, or alky- oraryloxycarbonyloxyalkyl halide. The reaction mixture is stirredovernight at a temperature between 40 to 100° C. After cooling to roomtemperature, insolubles can optionally be filtered off and the reactionmixture diluted with one molar (1.0 M) hydrochloric acid (HCl) and anappropriate organic solvent such as methyl tert-butyl ether (MTBE),diethyl ether (Et₂O), ethylacetate (EtOAc), or mixtures thereof. Afterphase separation, the aqueous phase is extracted several times with thesame solvent. The combined organic extracts are washed with water,brine, and dried over anhydrous magnesium sulfate (MgSO₄). Afterfiltration, the organic solvents are removed under reduced pressureusing a rotary evaporator. If required, the crude reaction products arefurther purified by well known purification techniques such as silicagel flash column chromatography (i.e., Biotage), mass-guidedreversed-phase preparative HPLC/lyophilization, precipitation, orcrystallization.

General Procedure B1: Activation of Carboxylic Acid Derivatives withDehydration Agents for Aminolysis or Alcoholysis

(2E)-3-(Methoxycarbonyl)prop-2-enoic acid (methyl hydrogen fumarate,MHF), 2-[(2E)-3-(methoxycarbonyl) prop-2-enoyloxy]acetic acid (23) or2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24), (1.0equivalents) are reacted at temperature from ca. 0° C. (ice bath) toroom temperature with 1.0-1.5 equivalents of a carbodiimide dehydrationagent such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC,EDC), N,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide(DCC) in an inert solvent such as dichloromethane (DCM),N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), orN,N-dimethylacetamide (DMA, DMAc) (ca. 3 mL/mmol). 1.0-1.5 Equivalentsof an appropriately functionalized amine or 2-hydroxy acetamidedissolved in the same solvent and, optionally, in the presence of acatalytic or stoichiometric amount of 4-(N,N-dimethylaminopyridine(DMAP) is added at a temperature from ca. 0° C. to room temperature.When the amine is a salt form, an equimolar amount of an organictertiary base, such as triethylamine (TEA), or diisopropylethylamine(DIEA) may be added to free the amine base prior to the coupling step.The reaction mixture is stirred for 4 to 12 hours at room temperature.Optionally the organic solvents are removed under reduced pressure usinga rotary evaporator and the residue diluted with an appropriateextraction solvent such as diethyl ether (Et₂O), methyl tert-butyl ether(MTBE), ethyl acetate (EtOAc), or others. The procedures described inProcedure A for product isolation and purification may be employed.

General Procedure B2: Activation of Carboxylic Acid Derivatives withChlorination Agents and Aminolysis

2-[(2E)-3-(Methoxycarbonyl) prop-2-enoyloxy]acetic acid (23) or2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24) (1.0equivalents) are reacted with oxalyl chloride (1.0-1.5 equivalents) inanhydrous dichloromethane (DCM), ca. 3 mL/mmol, at a temperature of ca.0° C. (ice bath) in the presence of a catalytic amount ofN,N-dimethylformamide (DMF) for 1 to 3 hours. The solvents are removedunder reduced pressure using a rotary evaporator and the crude materialis dissolved in anhydrous dichloromethane (DCM), ca. 3 mL/mmol. 1.0-1.5Equivalents of an appropriately functionalized nucleophile (primary orsecondary amine, or alcohol) in anhydrous dichloromethane (DCM), ca. 3mL/mmol, are added dropwise at ca. 0° C. (ice bath), optionally in thepresence of a catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP).When the amine component is a salt form, an equimolar amount of a base,such as triethyamine (TEA), diisopropylethylamine (DIEA), or others, areadded to free the amine base prior to the coupling step. The reaction isstirred overnight with warming to room temperature, the solventsoptionally removed under reduced pressure using a rotary evaporator, andthen diluted with an appropriate extraction solvent such as diethylether (Et₂O), methyl tert-butyl ether (MTBE), ethyl acetate (EtOAc), orothers. The procedures described in Procedure A for product isolationand purification may be employed.

Example 1 (N,N-Diethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate(1)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.39 g,3.00 mmol) dissolved in NMP was reacted at ca. 55° C. with2-chloro-N,N-diethylacetamide (0.44 g, 3.00 mmol) in the presence ofCsHCO₃ (0.69 g, 3.60 mmol) to afford 0.37 g (51% yield) of the titlecompound (1) after purification by silica gel column chromatography(Biotage) using a mixture of ethyl acetate (EtOAc) and hexanes (1:1) aseluent. M.p.: 53-56° C. ¹H NMR (CDCl₃, 400 MHz): δ 6.99-6.90 (m, 2H),4.83 (s, 2H), 3.80 (s, 3H), 3.39 (q, J=7.2 Hz, 2H), 3.26 (q, J=7.2 Hz,2H), 1.24 (t, J=7.2 Hz, 3H), 1.14 (t, J=7.2 Hz, 3H). MS (ESI): m/z244.13 (M+H)⁺.

Example 2 Methyl [N-benzylcarbamoyl]methyl(2E)but-2-ene-1,4-dioate (2)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.85 mmol) dissolved in NMP was reacted at ca. 55° C. with N-benzylchloroacetamide (0.84 g, 4.61 mmol) in the presence of CsHCO₃ (0.89 g,4.61 mmol) to afford 0.56 g (53% yield) of the title compound (2) as awhite solid after purification by mass-guided preparative HPLC andlyophilization. ¹H NMR (CDCl₃, 400 MHz): δ 7.36-7.26 (m, 5H), 6.94-6.88(m, 2H), 6.19 (br s, 1H), 4.73 (s, 2H), 4.51 (d, J=5.6 Hz, 2H), 3.81 (s,3H). MS (ESI): m/z 278.04 (M+H)⁺.

Example 3 Methyl 2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate (3)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with4-(chloroacetyl) morpholine (0.75 g, 4.61 mmol) in the presence ofCsHCO₃ (0.89 g, 4.61 mmol) to afford 0.34 g (35% yield) of the titlecompound (3) as a white solid after purification by mass-guidedpreparative HPLC and lyophilization. M.p.: 124 to 126° C.; ¹H NMR(CDCl₃, 400 MHz): δ 6.97-6.91 (m, 2H), 4.84 (s, 2H), 3.82 (s, 3H),3.72-3.70 (m, 4H), 3.64-3.62 (m, 2H), 3.46-3.41 (m, 2H). MS (ESI): m/z258.04 (M+H)⁺.

Example 4 (N-Butylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate (4)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with N-butylchloroacetamide (0.69 g, 4.61 mmol) in the presence of CsHCO₃ (0.89 g,4.61 mmol) to afford 0.19 g (21% yield) of the title compound (4) as awhite solid after purification by mass-guided preparative HPLC andlyophilization. ¹H NMR (CDCl₃, 400 MHz): δ 6.98-6.92 (m, 2H), 6.09 (brs, 1H), 4.68 (s, 2H), 3.82 (s, 3H), 3.34-3.29 (q, 2H, J=6.4 Hz),1.54-1.48 (m, 2H), 1.38-1.32 (m, 2H), 0.956-0.920 (t, J=7.6 Hz, 3H). MS(ESI): m/z 244.04 (M+H)⁺.

Example 5 [N-(2-Methoxyethyl)carbamoyl]methylmethyl(2E)but-2-ene-1,4-dioate (5)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. withN-(2-methoxyethyl) chloroacetamide (0.69 g, 4.60 mmol) in the presenceof CsHCO₃ (0.89 g, 4.61 mmol) to afford 0.07 g (8% yield) of the titlecompound (5) as a white solid after purification by mass-guidedpreparative HPLC and lyophilization. ¹H NMR (CDCl₃, 400 MHz): δ6.94-6.92 (m, 2H), 6.46 (br s, 1H), 4.68 (s, 2H), 3.83 (s, 3H),3.52-3.46 (m, 4H), 3.36 (s, 3H). MS (ESI): m/z 245.98 (M+H)⁺.

Example 62-{2-[(2E)-3-(Methoxycarbonyl)prop-2-enoyloxy]acetylamino}acetic acid(6)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.68 g,5.26 mmol) dissolved in NMP was reacted at ca. 55° C. with tert-butyl2-(2-chloroacetylamino) acetate (0.91 g, 4.38 mmol) in the presence ofCsHCO₃ (1.19 g, 6.13 mmol) of the tert-butyl-protected intermediate andthen purified by silica gel column chromatography (Biotage) using amixture of ethyl acetate (EtOAc) and hexanes (1:2 to 2:3 to 1:1) aseluent. The purified product was treated with 50% trifluoroacetic acid(TFA) in dichloromethane (DCM). Removal of solvents afforded 0.13 g (12%yield) of the title compound (6). ¹H NMR (CD₃OD, 400 MHz): δ 6.96-6.93(m, 2H), 4.74 (s, 2H), 3.98-3.95 (m, 2H), 3.81 (s, 3H). MS (ESI): m/z246.00 (M+H)⁺, 244.02 (M−H)⁻.

Example 74-{2-[(2E)-3-(Methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid(7)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.56 g,4.33 mmol) dissolved in NMP was reacted at ca. 55° C. with tert-butyl4-(2-chloroacetylamino) butanoate (0.85 g, 3.61 mmol) in the presence ofCsHCO₃ (0.98 g, 5.05 mmol) of the tert-butyl-protected intermediate andthen purified by silica gel column chromatography (Biotage) using amixture of ethyl acetate (EtOAc) and hexanes (1:1) as eluent. Thepurified product was treated with 50% trifluoroacetic acid (TFA) indichloromethane (DCM). Removal of solvents afforded 0.45 g (46% yield)of the title compound (7). ¹H NMR (CD₃OD, 400 MHz): δ 6.94-6.91 (m, 2H),4.65 (s, 2H), 3.81 (s, 3H), 3.28 (t, J=6.8 Hz, 2H), 2.33 (t, J=7.2 Hz,2H), 1.81 (p, J=7.1 Hz, 2H). MS (ESI): m/z 274.03 (M+H)⁺ 272.06 (M−H)⁻.

Example 8Methyl(N-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl(2E)but-2ene-1,4-dioate(8)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with2-chloro-N-1,3,4-thiadiazol-2-ylacetamide (0.81 g, 4.61 mmol) in thepresence of CsHCO₃ (0.89 g, 4.61 mmol). The crude material precipitatedout and was washed several times with dichloromethane (DCM) for furtherpurification to afford 0.12 g (12% yield) of the title compound (8) as awhite solid. ¹H NMR (CD₃OD, 400 MHz): δ 9.06 (s, 1H), 6.95-6.91 (m, 2H),4.99 (s, 2H), 3.82 (s, 3H). MS (ESI): m/z 272.07 (M+H)+

Example 9 N,N-Dimethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate(9)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with N,N-dimethylchloroacetamide (0.56 g, 4.61 mmol) in the presence of CsHCO₃ (0.89 g,4.61 mmol). The crude material was precipitated out from a mixture ofethyl acetate (EtOAc) and hexanes (Hxn) (1:1) to provide a white solid.This solid was further dissolved in dichloromethane (DCM) and theorganic layer washed with water. After removal of the solvents 0.55 g(67% yield) of the title compound (9) was obtained as a white solid. ¹HNMR (CDCl₃, 400 MHz): δ 6.98-6.90 (m, 2H), 4.84 (s, 2H), 3.80 (s, 3H),2.99-2.97 (2s, 6H). MS (ESI): m/z 216 (M+H)⁺.

Example 10 (N-Methoxy-N-methylcarbamoyl)methylmethyl(2E)but-2-ene-1,4-dioate (10)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with N-methyl,N-hydroxymethyl chloroacetamide (0.63 g, 4.61 mmol) in the presence ofCsHCO₃ (0.89 g, 4.61 mmol). The crude material precipitated out from aconcentrated ethyl acetate (EtOAc) solution. The solid was filtered offand dried in vacuum to provide 0.54 g (61% yield) of the title compound(10) as a white solid. ¹H NMR (CD₃OD, 400 MHz): δ 6.92-6.89 (m, 2H),5.01 (s, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.20 (s, 3H). MS (ESI): m/z232.06 (M+H)⁺.

Example 11 bis-(2-Methoxyethylamino)carbamoyl]methylmethyl(2E)but-2-ene-1,4-dioate (11)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. withbis-(2-methoxyethyl)-chloroacetamide (0.96 g, 4.61 mmol) in the presenceof CsHCO₃ (0.89 g, 4.61 mmol) to afford 0.53 g (46% yield) of the titlecompound (11) as a white solid after purification by mass-guidedpreparative HPLC and lyophilization. M.p.: 79-82° C.; ¹H NMR (CDCl₃, 400MHz): δ 6.98-6.88 (m, 2H), 4.98 (s, 2H), 3.8 (s, 3H), 3.57-3.50 (m, 8H),3.41 (s, 3H), 3.31 (s, 3H). MS (ESI): m/z 304.14 (M+H)⁺.

Example 12 [N-(Methoxycarbonyl)carbamoyl]methylmethyl(2E)but-2ene-1,4-dioate (12)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. withmethyl-N-(2-chloroacetyl) carbamate (0.69 g, 4.61 mmol) in the presenceof CsHCO₃ (0.89 g, 4.61 mmol). The crude material precipitated out froma diethyl ether (Et₂O) solution. The solid was filtered off, washedseveral times with dichloromethane (DCM), and dried in vacuum to provide0.19 g (21% yield) of the title compound (12) as a white solid. ¹H NMR(CDCl₃, 400 MHz): δ 6.99-6.91 (m, 2H), 5.23 (s, 2H), 3.81 (s, 6H). MS(ESI): m/z 246.09 (M+H)⁺, 268.00 (M+Na⁺)⁺.

Example 13[N-(2-{2-[(2E)-3-(Methoxycarbonyl)prop-2-enoyloxy]acetylamino}ethyl)carbamoyl]methylmethyl(2E)but-2ene-1,4-dioate (13)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.73 g,5.61 mmol) dissolved in NMP was reacted at ca. 55° C. withN-(2-[(2-chloroacetyl]aminoethyl)-2-chloroactamide (0.50 g, 2.34 mmol)in the presence of CsHCO₃ (1.08 g, 5.61 mmol). The crude materialprecipitated out from a concentrated diethyl ether (Et₂O) solution. Thesolid was filtered off, washed several times with dichloromethane (DCM),and dried in vacuum to provide 0.90 g (96% yield) of the title compound(13) as a white solid. ¹H NMR (400 MHz, DMSO-d⁶): δ 8.23 (br s, 2H),6.96-6.92 (m, 4H), 4.58 (s, 4H), 3.79 (s, 6H), 3.16 (m, 4H). MS (ESI):m/z 401.05 (M+H)⁺.

Example 14 Methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioateHydrochloride (14)

Following general procedure A, methyl hydrogen fumarate (MHF) (1.00 g,7.68 mmol) dissolved in NMP was reacted at ca. 55° C. with1-(tert-butyloxycarbonyl)-4-chloroacetyl piperazine (2.42 g, 9.22 mmol)in the presence of CsHCO₃ (1.78 g, 9.22 mmol). After work-up and removalof the solvent, the crude material was obtained as a white solid. Thesolid was reacted at room temperature with 15 mL of a 4 molar (4 M)solution of hydrogen chloride (HCl) in 1,4-dioxane. After removal of thesolvents, the solid hydrochloride salt was further purified bymass-guided preparative HPLC to provide 0.93 g (41% yield) of the titlecompound (14) as a white solid after lyophilization of the solvents inthe presence of an excess of aqueous 1 normal (1 N) hydrochloric acid.¹H NMR (D₂O, 400 MHz): δ 6.93-6.86 (m, 2H), 4.92 (s, 2H), 3.70-3.63 (m,7H), 3.23 (t, J=5.2 Hz, 2H), 3.17 (t, J=6 Hz, 2H). MS (ESI): m/z 257.13(M+H)⁺.

Example 15 Methyl2-(4-benzylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate (15)

Methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate hydrochloride(14) (0.50 g, 1.71 mmol) was reacted at ca. 0° C. with benzyl bromide(BnBr) (0.243 mL, 0.35 g, 2.05 mmol) and diisopropylethylamine (DIEA)(1.00 mL, 0.74 g, 5.76 mmol) in dichloromethane (DCM) followed bywarming to room temperature. After aqueous work-up, the crude productwas purified by mass-guided preparative HPLC to afford 0.18 g (27%yield) of the title compound (15) as a white solid. ¹H NMR (CDCl₃, 400MHz): δ 7.08-7.01 (m, 5H), 6.72-6.71 (m, 2H), 4.60 (s, 2H), 3.58-3.57(s, 3H), 3.23-3.19 (br S, 2H), 3.30 (s, 2H), 3.1.19-3.11 (br S, 2H),2.23 (br S, 4H); MS (ESI) m/z 347.13 (M+H)⁺.

Example 16 2-(4-Acetylpiperazinyl)-2oxoethylmethyl(2E)but-2ene-1,4-dioate (16)

Methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate hydrochloride(14) (0.20 g, 0.68 mmol) was reacted with acetyl chloride (AcCl) (0.60mL, 0.66 g, 0.84 mmol) and diisopropylethylamine (0.70 mL, 0.52 g, 4.0mmol) in dichloromethane (DCM). Following aqueous work-up, the crudeproduct was purified by silica gel flash chromatography to afford 0.12 g(54% yield) of the title compound (16) as a white solid. ¹H NMR (CDCl₃,400 MHz): δ 6.98-6.93 (m, 2H), 4.86 (s, 2H), 3.83 (s, 3H), 3.66 3.63 (m,4H), 3.50-3.40 (m, 4H), 2.14 (s, 3H). MS (ESI): m/z 299.12 (M+H)⁺.

Example 17 Methyl2-oxo-2-(2-oxo(1,3-oxazolidin-3-yl)ethyl(2E)but-2ene-1,4-dioate (17)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with3-(chloroacetyl)-1,3-oxazolidine-2-one (0.75 g, 4.61 mmol) in thepresence of CsHCO₃ (0.89 g, 4.61 mmol) to yield 0.30 g (30% yield) ofthe title compound (17) as a white solid after purification bymass-guided preparative HPLC and lyophilization. ¹H NMR (CDCl₃, 400MHz): δ 6.97-6.92 (m, 2H), 5.32 (s, 2H), 4.53 (t, J=8 Hz, 2H), 4.05 (t,J=8.0 Hz, 2H), 3.82 (s, 3H). MS (ESI); m/z 258.20 (M+H)⁺.

Example 18 {N-[2-(Dimethylamino)ethyl]carbamoyl}methylmethyl(2E)but-2ene-1,4 dioate (18)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. withN,N-dimethylethylenediamino chloroacetamide (0.75 g, 4.61 mmol) in thepresence of CsHCO₃ (0.89 g, 4.61 mmol) to provide 0.02 g (2% yield) ofthe title compound (18) as a white solid after purification bymass-guided preparative HPLC and lyophilization. ¹H NMR (D₂O, 400 MHz):δ 8.27, (s, 1H), 6.87-6.78, (m, 2H), 4.63 (s, 2H), 3.68 (s, 3H), 3.51(t, J=6.2 Hz, 2H), 3.17 (t, J=6.0 Hz, 2H), 2.76 (s, 6H). MS (ESI); m/z259.14 (M+H)⁺.

Example 19 Methyl{N-[(propylamino)carbonyl]carbamoyl}methyl(2E)but-2ene-1,4-dioate (19)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with1-(2-chloroacetyl)-3-propyl urea (0.82 g, 4.60 mmol) in the presence ofCsHCO₃ (0.89 g, 4.61 mmol) to provide 0.02 g (2% yield) of the titlecompound (19) as a white solid. Addition of methanol (MeOH) afforded0.49 g (48% yield) of white solid. ¹H NMR (CDCl₃, 400 MHz): δ 6.90-6.99(m, 2H), 4.77 (s, 2H), 3.82 (s, 3H), 3.25-3.24 (q, 2H, J=5.6 Hz),1.57-1.55 (q, 2H, J=7.2 Hz), 0.95-0.91 (t, 3H, J=7.6 Hz). MS (ESI): m/z273.08 (M+H)⁺.

Example 20 2-{(2S)-2-[(tert-Butyl)oxycarbonyl]pyrrolidinyl}-2-oxoethylmethyl(2E)but-2ene-1,4-dioate (20)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. withtert-butyl(2S)-1-(2-chloroacetyl)pyrrolidine-2-carboxylate (0.82 g, 4.60mmol) in the presence of CsHCO₃ (0.89 g, 4.61 mmol) to provide 0.44 g(34% yield) of the title compound (20) as a white solid afterpurification by mass-guided preparative HPLC and lyophilization. ¹H NMR(CDCl₃, 400 MHz, all rotamers): δ 6.97-6.90 (m, 2H), 4.91-4.55 (m, 2H),4.44-4.29 (m, 1H), 3.80 (s, 3H), 3.61-3.58 (m, 2H), 2.23-2.03 (br m,4H), 1.54-1.46 (s, 9H). MS (ESI): m/z 342.16 (M+H)⁺, 364.09 (M+Na⁺)⁺.

Example 21 (N-{[tert-Butyl)oxycarbonyl]methyl}-N-methylcarbamoyl)methylmethyl (2E)but-2ene1,4-dioate (21)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with tert-butyl2-(2-chloro-N-methylacetylamino) acetate (1.02 g, 4.60 mmol) in thepresence of CsHCO₃ (0.89 g, 4.61 mmol) to provide 0.24 g (21% yield) ofthe title compound (21) as a white solid after purification by silicagel flash chromatography (Biotage). ¹H NMR (CDCl₃, 400 MHz, allrotamers): δ 7.00-6.93 (m, 2H), 4.90-4.79 (2s, 2H), 4.03-3.89 (2s, 2H),3.80 (s, 3H), 3.04-2.99 (2S, 3H), 1.45 (S, 9H). MS (ESI): m/z 316.13(M+H)⁺.

Example 22 {N-(Ethoxycarbonyl)methyl]-N-methylcarbamoyl}methylmethyl(2E)but-2-ene-1,4-dioate (22)

Following general procedure A, methyl hydrogen fumarate (MHF) (0.50 g,3.84 mmol) dissolved in NMP was reacted at ca. 55° C. with ethyl2-(2-chloro-N-methylacetylamino) acetate (0.89 g, 4.60 mmol) in thepresence of CsHCO₃ (0.89 g, 4.61 mmol) to provide 0.30 g (27% yield) ofthe title compound (22) as a white solid after purification by silicagel flash chromatography (Biotage). ¹H NMR (CDCl₃, 400 MHz, allrotamers): δ 7.00-6.93 (m, 2H), 4.90-4.79 (2s, 2H), 4.03-3.89 (2s, 2H),3.80 (s, 3H), 3.04-2.99 (2s, 3H), 1.45 (s, 9H). MS (ESI): m/z 316.13(M+H)⁺.

Example 23 2-[(2E)-3-(Methoxycarbonyl)prop-2-enoyloxy]acetic acid (23)

Following general procedure A, methyl hydrogen fumarate (MHF) (6.91 g,53.12 mmol) dissolved in NMP was reacted at ca. 55° C. with tert-butyl2-chloroacetic acid (9.48 mL, 10.0 g, 66.4 mmol) in the presence ofCsHCO₃ (15.41 g, 79.68 mmol) to provide 13.11 g (81% yield) of theintermediate ester as a white solid after precipitation from aconcentrated diethyl ether (Et₂O) solution. The material was ofsufficient purity to be used in the next step. ¹H NMR (CDCl₃, 400 MHz):δ 6.95-6.92 (m, 2H), 4.61 (s, 2H), 3.81 (s, 3H), 1.47 (s, 9H). Thematerial was dissolved in 50 mL of 50 vol.-% of trifluoroacetic acid(TFA) in dichloromethane (DCM) and reacted overnight at roomtemperature. After removal of the solvents, the crude material wasprecipitated from a mixture of acetone and hexanes (1:3) to afford 12.3g (92% yield) of the title compound (23) as a white solid. ¹H NMR(CDCl₃, 400 MHz): δ 7.02-6.90 (m, 2H), 4.79 (s, 2H), 3.82 (s, 3H). MS(ESI): m/z 189.07 (M+H)⁺. MS (ESI): m/z 189.07 (M+H)⁺.

Example 24 rac-2-[(2E)-3-(Methoxycarbonyl)prop-2-enoyloxy]propanoic acid(24)

Following general procedure A, methyl hydrogen fumarate (MHF) (4.68 g,36.0 mmol) dissolved in NMP was reacted at ca. 55° C. withrac-tert-butyl 2-bromo propionic acid (4.98 mL, 6.27 g, 30.0 mmol) inthe presence of CsHCO₃ (6.40 g, 33.0 mmol) to yield the intermediateester. The material was of sufficient purity to be used in the nextstep. ¹H NMR (CDCl₃, 400 MHz): δ 6.93-6.88 (m, 2H), 5.02 (q, J=7.2 Hz,2H), 3.79 (s, 3H), 1.49 (d, J=7.2 Hz, 3H), 1.44 (s, 9H). The materialwas dissolved in 25 mL of 50 vol.-% of trifluoroacetic acid (TFA) indichloromethane (DCM) and reacted overnight at room temperature. Afterremoval of the solvents, the title compound (24) was obtained as a whitesolid that was of sufficient purity to be used in subsequent steps. ¹HNMR (CDCl₃, 400 MHz): δ 6.97-6.92 (m, 2H), 5.22 (q, J=7.2 Hz, 2H), 3.82(s, 3H), 1.60 (d, J=7.2 Hz, 3H).

Example 25 Methyl2-(4-methylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate (25)

Following general procedureB2,2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) (0.50 g,2.65 mmol) was activated withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC)(0.60 g, 3.18 mmol) in 10 mL of dichloromethane (DCM) at ca. 0° C.N-Methyl piperazine (0.353 mL, 0.31 g, 3.18 mmol) and4-(N,N-dimethyl)aminopyridine (DMAP) (0.40 g, 3.18 mmol) were added tothe activated carboxylic acid. After work-up and isolation, andpurification by mass-guided preparative HPLC afforded 0.09 g (13% yield)of the title compound (25) as a white solid after lyophilization. ¹H NMR(CDCl₃, 400 MHz): δ 6.93-6.78 (m, 2H), 4.77 (s, 2H), 3.73 (s, 3H),3.56-3.54 (m, 2H), 3.35-3.25 (m, 2H), 2.37-2.33 (m, 4H), 2.31 (s, 3H).MS (ESI): m/z 271.13 (M+H)⁺.

Example 26 {N,N-bis[2-(Methylethoxyethyl]carbamoyl}methylmethyl(2E)but-2-ene-1,4-dioate (26)

Following general procedure B2,2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) (0.50 g, 2.65 mmol) was activated withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC)(0.60 g, 3.18 mmol) in 10 mL of dichloromethane (DCM) at ca. 0° C.bis(2-Isopropoxyethyl)amine (0.60 g, 3.18 mmol) and4-(N,N-dimethyl)aminopyridine (DMAP) (0.40 g, 3.18 mmol) were added tothe activated carboxylic acid. After work-up and isolation, andpurification by silica gel flash column chromatography (Biotage) usingethyl acetate (EtOAc) and hexanes (1:1) afforded 0.30 g (32% yield) ofthe title compound (26) as a white solid following refrigeration. ¹H NMR(CDCl₃, 400 MHz): δ 6.95-6.86 (m, 2H), 4.98 (s, 2H), 3.76 (s, 3H),3.50-3.47 (m, 10H), 1.10-1.05 (m, 12H). MS (ESI): m/z 360.16 (M+H)⁺.

Example 27 [N,N-bis(2-Ethoxyethyl)carbamoyl]methylmethyl(2E)but-2-ene-1,4-dioate (27)

Following general procedure B2,2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) (0.80 g, 6.14 mmol) was activated withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC)(1.40 g, 7.37 mmol) in 20 mL of dichloromethane (DCM) at ca. 0° C.bis(2-Ethoxyethyl)amine hydrochloride (1.18 g, 7.37 mmol) (1.18 g, 7.37mmol) and diisopropylethylamine (DIEA) (1.34 mL, 0.99 g, 7.67 mmol) wereadded to the activated carboxylic acid. After work-up and isolation, andpurification by silica gel flash column chromatography (Biotage) usingethyl acetate (EtOAc) and hexanes (1:1) afforded 0.30 g (15% yield) ofthe title compound (27) as a white solid. ¹H NMR (CDCl₃, 400 MHz): δ6.97 (d, J=15.6 Hz, 1H), 6.94 (d, J=1.6 Hz, 1H), 5.01 (s, 2H), 3.80 (s,3H), 3.56-3.43 (m, 12H), 1.19 (q, J=7.6 Hz, 6H). MS (ESI): m/z 332.20(M+H)⁺.

Example 28 Methyl1-methyl-2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate

Following general procedure B2, 2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24) (0.48 g, 2.40 mmol) was activatedwith N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC)(0.64 g, 3.36 mmol) in 10 mL of dichloromethane (DCM) at ca. 0° C.Morpholine (0.25 mL, 0.25 g, 2.88 mmol) was added to the activatedcarboxylic acid. After work-up and isolation, and purification bymass-guided preparative HPLC afforded 0.22 g (33% yield) of the titlecompound (28) as a white solid after lyophilization. M.p.: 70-73° C. ¹HNMR (CDCl₃, 400 MHz): δ 6.95-6.89 (m, 2H), 5.45 (q, J=6.8 Hz, 1H), 3.80(s, 3H), 3.71-3.68 (m, 4H), 3.58-3.54 (m, 4H), 1.48 (d, J=7.2 Hz, 3H).MS (ESI): m/z 272.13 (M+H)⁺.

Example 29 [N,N-bis(2-Methoxyethyl)carbamoyl]ethylmethyl(2E)but-2-ene-1,4-dioate (29)

Following general procedure B2, 2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24) (0.48 g, 2.40 mmol) was activatedwith N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC)(0.64 g, 3.36 mmol) in 10 mL of dichloromethane (DCM) at ca. 0° C.bis(2-Methoxyethyl)amine (0.42 mL, 0.37 g, 2.88 mmol) was added to theactivated carboxylic acid. After work-up and isolation, and purificationby mass-guided preparative HPLC afforded 0.29 g (38% yield) of the titlecompound (29) as a white solid after lyophilization. ¹H NMR (CDCl₃, 400MHz): δ 6.94-6.88 (m, 2H), 5.52 (q, J=6.8 Hz, 1H), 3.80-3.79 (s, 3H),3.57-3.49 (m, 8H), 3.33-3.31 (2s, 6H), 1.48 (d, J=6.4 Hz, 3H). MS (ESI):m/z 318.13 (M+H)⁺.

Example 30 (N,N-Dimethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate(30)

Following general procedure B2, 2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24) (0.48 g, 2.40 mmol) was activatedwith N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC)(0.64 g, 3.36 mmol) in 10 mL of dichloromethane (DCM) at ca. 0° C.N,N-Dimethylamine hydrochloride (0.23 g, 2.88 mmol) anddiisopropylethylamine (DIEA) (0.63 mL, 0.467 g, 3.61 mmol) were added tothe activated carboxylic acid. After work-up and isolation, andpurification by mass-guided preparative HPLC afforded 0.25 g (46% yield)of the title compound (30) as a white solid after lyophilization. ¹H NMR(CDCl₃, 400 MHz): δ 6.93-6.86 (m, 2H), 5.46 (q, J=6.8 Hz 1H), 3.79 (s,3H), 3.06-2.97 (2s, 6H), 1.47 (d, J=6.4 Hz, 3H). MS (ESI): m/z 230.13(M+H)⁺.

Example 31 (1S)-1-Methyl-2-morpholin-4-yl-2-oxoethylmethyl(2E)but-2-ene-1,4-dioate (31)

Following general procedure B2, (2E)-3-(methoxycarbonyl)prop-2-enoicacid (methyl hydrogen fumarate, MHF) (0.50 g, 3.84 mmol) was activatedwith N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC)(0.81 g, 4.20 mmol) in 10 mL of dichloromethane (DCM) at ca. 0° C.(2S)-2-Hydroxy-1-morpholin-4-yl-propan-1-one (0.48 g, 3.07 mmol) and4-(N,N-dimethyl)aminopyridine (DMAP) (0.40 g, 3.18 mmol) were added tothe activated carboxylic acid. After work-up and isolation, andpurification by silica gel flash column chromatography (Biotage) usingethyl acetate (EtOAc) and hexanes (ca. 3:2) afforded 0.42 g (51% yield)of the title compound (31) as a white solid. M.p.: 79-82° C.; ¹H NMR(CD₃CN, 400 MHz): δ 6.90-6.81 (m, 2H), 5.44 (q, J=6.8 Hz 1H), 3.78 (s,3H), 3.65-3.60 (m, 4H), 3.51-3.50 (m, 4H), 1.42 (d, J=6.8 Hz 3H). MS(ESI): m/z 272.05 (M+H)⁺.

Example 32 (1S)-1-[N,N-bis(2-Methoxyethyl)carbamoyl]ethylmethyl(2E)but-2-ene-1,4-dioate (32)

Following general procedure B2, (2E)-3-(methoxycarbonyl)prop-2-enoicacid (methyl hydrogen fumarate, MHF) (0.50 g, 3.84 mmol) was activatedwith N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC)(0.88 g, 4.60 mmol) in 20 mL of dichloromethane (DCM) at ca. 0° C.(2S)-2-Hydroxy-N,N-bis(2-methoxyethyl)propanamide (0.63 g, 3.07 mmol)and 4-(N,N-dimethyl)aminopyridine (DMAP) (0.40 g, 3.18 mmol) were addedto the activated carboxylic acid. After work-up and isolation, andpurification by silica gel flash column chromatography (Biotage) usingethyl acetate (EtOAc) and hexanes (2:1) afforded 0.16 g (14% yield) ofthe title compound (32) as a clear oil. ¹H NMR (CDCl₃, 400 MHz): δ6.93-6.77 (m, 2H), 5.53 (q, J=6.4 Hz, 1H), 3.80 (s, 3H), 3.58-3.50 (m,8H), 3.47-3.32 (2s, 6H), 1.49 (d, J=6.8 Hz, 3H). MS (ESI): m/z 318.05(M+H)⁺.

Example 33 (1S)-1-(N,N-Diethylcarbamoyl)ethylmethyl(2E)but-2-ene-1,4-dioate (33)

Following general procedure B2, (2E)-3-(methoxycarbonyl)prop-2-enoicacid (methyl hydrogen fumarate, MHF) (0.50 g, 3.84 mmol) was activatedwith N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC)(0.88 g, 4.60 mmol) in 12 mL of dichloromethane (DCM) at ca. 0° C.(2S)—N,N-Diethyl-2-hydroxypropanamide (0.44 g, 3.07 mmol) (0.44 g, 3.07mmol) and 4-(N,N-dimethyl)aminopyridine (DMAP) (0.40 g, 3.18 mmol) wereadded to the activated carboxylic acid. After work-up and isolation, andpurification by mass-guided preparative HPLC/lyophilization and bysilica gel flash column chromatography (Biotage) using ethyl acetate(EtOAc) and hexanes afforded 0.17 g (18% yield) of the title compound(33) as a clear oil. ¹H NMR (CDCl₃, 400 MHz): δ 6.95-6.87 (m, 2H), 5.43(q, J=6.8 Hz, 1H) 3.80 (s, 3H), 3.50-3.26 (m, 4H), 1.49 (d, J=6.4 Hz,3H), 1.26 (t, J=6.8, 3H), 1.12 (t, J=7.6 Hz, 3H). MS (ESI): m/z 258.06(M+H)⁺.

Example 34 (N-{[(tert-Butyl)oxycarbonyl]methyl}carbamoyl)methylmethyl(2E)but-2-ene-1,4-dioate (34)

Following general procedure B2, 2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) (0.50 g, 2.65 mmol) was activated withoxalyl chloride (0.30 mL, 0.40 g, 3.18 mmol) in dichloromethane (DCM) atca. 0° C. and in the presence of a catalytic amount ofN,N-dimethylformamide (DMF). A DCM solution of the freshly preparedcrude acid chloride was reacted ca. 0° C. (ice bath) with glycinetert-butyl ester (H-GlyOtBu) (0.53 g, 3.18 mmol) in DCM and in thepresence of 4-(N,N-dimethyl)aminopyridine (DMAP) (0.40 g, 3.18 mmol).After aqueous work-up and isolation, and purification by silica gelflash chromatography afforded 0.16 g (20% yield) of the title compound(34) as a semi-solid material. ¹H NMR (CDCl₃, 400 MHz, all rotamers): δ6.95-6.69 (m, 2H), 6.63 (br. m, 1H), 4.73 (s, 2H), 3.99 (d, J=4.8 Hz,2H), 3.83 (s, 3H), 1.48 (s, 9H). MS (ESI): m/z 324.05 (M+Na⁺)⁺.

Example 35Methyl(N-methyl-N-{[(methylethyl)oxycarbonyl]methyl}carbamoyl)methyl(2E)but-2-ene-1,4-dioate (35)

Following general procedure B2, 2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) (0.50 g, 2.65 mmol) was activated withoxalyl chloride (0.30 mL, 0.40 g, 3.18 mmol) in dichloromethane (DCM) atca. 0° C. and in the presence of a catalytic amount ofN,N-dimethylformamide (DMF). A DCM solution of the freshly preparedcrude acid chloride was reacted ca. 0° C. (ice bath) with sarcosineisopropyl ester (H-Sar-OiPr) (0.41 g, 3.18 mmol) anddiisopropylethylamine (DIEA) (0.41 mL, 0.304 g, 2.35 mmol) in DCM and inthe presence of 4-(N,N-dimethyl)aminopyridine (DMAP) (0.10 g, 0.82mmol). After aqueous work-up and isolation, and purification by silicagel flash chromatography afforded 0.214 g (27% yield) of the titlecompound (35) as a pale-yellow solid. ¹H NMR (CDCl₃, 400 MHz, allrotamers): δ 6.94-6.90 (m, 2H), 5.09-4.99 (m, 1H), 4.89-4.79 (2s, 2H),4.07-3.95 (2s, 2H), 3.78 (s, 3H), 3.04-2.98 (2s, 3H), 1.27-1.21 (m, 6H).MS (ESI): m/z 302.04 (M+H)⁺.

Example 36 {N-[(Ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methylmethyl(2E)but-2-ene-1,4-dioate (36)

Following general procedure B2, 2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) (0.50 g, 2.65 mmol) was activated withoxalyl chloride (0.27 mL, 0.40 g, 3.15 mmol) in dichloromethane (DCM) atca. 0° C. and in the presence of a catalytic amount ofN,N-dimethylformamide (DMF). A DCM solution of the freshly preparedcrude acid chloride was reacted ca. 0° C. (ice bath) with N-benzylglycine ethyl ester (Bn-Gly-OEt) (0.61 g, 3.18 mmol) in DCM and anexcess of diisopropylethylamine (DIEA) in the presence of a catalyticamount of 4-(N,N-dimethyl)aminopyridine (DMAP). After aqueous work-upand isolation, and purification by silica gel flash chromatographyafforded 0.12 g (13% yield) of the title compound (36) as a white solid.¹H NMR (CDCl₃, 400 MHz, all rotamers): δ 7.37-7.20 (m, 5H), 6.97-6.86(m, 2H), 4.94-4.83 (2s, 2H), 4.63-4.55 (2s, 2H), 4.18-4.14 (m, 2H),4.04-3.88 (2s, 2H), 3.79 (s, 3H), 1.24-1.20 (m, 3H). MS (ESI): m/z364.15 (M+H)⁺.

Example 37 {N-[(Ethoxycarbonyl)methyl]-N-benzylcarbamoyl}ethylmethyl(2E)but-2-ene-1,4-dioate (37)

Following general procedure B2,2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24) (0.50 g,2.47 mmol) was activated with oxalyl chloride (0.25 mL, 0.35 g, 2.71mmol) in dichloromethane (DCM) at ca. 0° C. and in the presence of acatalytic amount of N,N-dimethylformamide (DMF). A DCM solution of thefreshly prepared crude acid chloride was reacted ca. 0° C. (ice bath)with N-benzyl glycine ethyl ester (Bn-Gly-OEt) (0.56 g, 2.90 mmol) inDCM and diisopropylethylamine (DIEA) (0.506 mL, 0.376 g, 2.90 mmol) inthe presence of a catalytic amount of 4-(N,N-dimethyl)aminopyridine(DMAP). After aqueous work-up and isolation, and purification by silicagel flash chromatography afforded 0.31 g (33% yield) of the titlecompound (37) as a white solid. ¹H NMR (CDCl₃, 400 MHz, all rotamers): δ7.37-7.17 (m, 5H), 6.88-6.77 (m, 2H), 5.49 (q, J=6.4 Hz, 0.75H), 5.33(q, J=6.4 Hz, 0.25H), 4.7-4.27 (m, 3H), 4.16-4.13 (m, 2H), 3.83-3.63 (m,4H), 1.53 (d, J=6.8 Hz, 3H), 1.21 (t, J=4.0 Hz, 3H). MS (ESI): m/z378.10 (M+H)⁺.

Example 38 {N-[(Ethoxycarbonyl)methyl]-N-methylcarbamoyl}ethyl methy(2E)but-2-ene-1,4-dioate (38)

Following general procedure B2,2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]propanoic acid (24) (0.50 g,2.47 mmol) was activated with oxalyl chloride (0.25 mL, 0.35 g, 2.71mmol) in dichloromethane (DCM) at ca. 0° C. and in the presence of acatalytic amount of N,N-dimethylformamide (DMF). A DCM solution of thefreshly prepared crude acid chloride was reacted ca. 0° C. (ice bath)with sarcosine ethyl ester (H-Sar-OEt) (0.43 g, 2.90 mmol) anddiisopropylethylamine (DIEA) (0.506 mL, 0.376 g, 2.90 mmol) in DCM andin the presence of a catalytic amount of 4-(N,N-dimethyl)aminopyridine(DMAP). After aqueous work-up and isolation, and purification by silicagel flash chromatography afforded 0.30 g (39% yield) of the titlecompound (38) as a white solid. ¹H NMR (CDCl₃, 400 MHz, all rotamers): δ6.88-6.81 (m, 2H), 5.47 (q, 0.75H, J=6.8 Hz), 5.32 (q, 0.25H, J=6.8 Hz),4.40-4.33 (m, 1H), 4.16-4.11 (m, 2H), 3.94-3.75 (m, 4H), 3.10 (s,2.25H), 2.96 (s, 0.75H), 1.50-1.44 (dd, 3H), 1.26-1.20 (m, 3H). MS(ESI): m/z 302.09 (M+H)⁺.

Example 39 Ethoxycarbonyloxyethyl methyl(2E)but-2-ene-1,4-dioate (39)

Following general procedure A, methyl hydrogen fumarate (0.39 g, 3.0mmol) in NMP (8 mL) was reacted with CsHCO₃ (0.69 g, 3.6 mmol) and ethyl(chloroethoxy)formate (0.64 g, 4.2 mmol) to afford 0.63 g (85% yield) ofthe title compound (39) after isolation and purification. ¹H NMR (CDCl₃,400 MHz): δ 6.89 (d, J=15.6 Hz, 1H), 6.82 (d, J=15.6 Hz, 1H), 6.84 (q,J=5.6 Hz, 1H), 4.23 (q, J=7.2 Hz, 3H), 3.81 (s, 3H), 1.58 (d, J=5.6 Hz,3H), 1.32 (t, J=7.2 Hz, 3H). MS (ESI): m/z 247.01 (M+H)⁺.

Example 40 Methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene-1,4-dioate(40)

Following general procedure A, methyl hydrogen fumarate (0.39 g, 3.0mmol) in NMP (8 mL) was reacted with CsHCO₃ (0.69 g, 3.6 mmol) andmethylethyl (chloroethoxy)formate (0.70 g, 4.2 mmol) to afford 0.71 g(91% yield) of the title compound (40) after isolation and purification.¹H NMR (CDCl₃, 400 MHz): δ 6.89 (d, J=15.6 Hz, 1H), 6.84 (q, J=5.2 Hz,1H, superimposed), 6.82 (d, J=15.6 Hz, 1H, superimposed), 4.90 (heptet,J=6.2 Hz, 3H), 3.81 (s, 3H), 1.57 (d, J=5.2 Hz, 3H), 1.32 (d, J=6.2 Hz,3H), 1.31 (d, J=6.2 Hz, 3H). MS (ESI): m/z 261.02 (M+H)⁺.

Example 41 (Cyclohexyloxycarbonyloxy)ethylmethyl(2E)but-2-ene-1,4-dioate (41)

Following general procedure A, methyl hydrogen fumarate (0.50 g, 3.85mmol) was reacted with CsHCO₃ (1.1 g, 5.71 mmol) and chloroethylcyclohexanecarboxylate (1.03 g, 4.99 mmol) at ca. 55° C. to afford 0.94g (82% yield) of the title compound (41) after purification bymass-guided preparatory HPLC and lyophilization. ¹H NMR (CDCl₃, 400MHz): δ 6.91-6.79 (m, 3H), 4.67-4.62 (m, 1H), 3.81 (s, 3H), 1.94-1.91(m, 2H), 1.77-1.73 (m, 2H), 1.57 (d, J=5.6 Hz, 3H), 1.53-1.46 (m, 3H),1.39-1.33 (m, 3H). MS (ESI); m/z 301.10 (M+H)⁺.

Example 42 Methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene-1,4-dioate(42)

Following general procedure A, methyl hydrogen fumarate (0.39 g, 3.0mmol) in NMP (8 mL) was reacted with CsHCO₃ (0.69 g, 3.6 mmol) andchloroethyl 2-methylpropanoate (0.63 g, 4.2 mmol) to afford 0.65 g (89%yield) of the title compound (42) after isolation and purification. ¹HNMR (CDCl₃, 400 MHz): δ 6.93 (q, J=5.2 Hz, 1H), 6.88 (d, J=16.0 Hz, 1H),6.81 (d, J=15.6 Hz, 1H), 3.81 (s, 3H), 2.55 (heptet, J=6.8 Hz, 3H), 1.54(d, J=5.2 Hz, 3H), 1.18 (d, J=6.8 Hz, 6H). MS (ESI): m/z 245.05 (M+H)⁺.

Example 43 Methyl phenylcarbonyloxyethyl(2E)but-2-ene-1,4-dioate (43)

Following general procedure A, methyl hydrogen fumarate (0.42 g, 3.3mmol) in NMP (6 mL) was reacted with CsHCO₃ (0.69 g, 3.6 mmol) andchloroethyl benzoate (0.55 g, 3.0 mmol) to afford 0.2 g (24% yield) ofthe title compound (43) after purification by silica gel flash columnchromatography (Biotage) using a mixture of ethyl acetate (EtOAc) andhexanes (1:8) as eluent. ¹H NMR (CDCl₃, 400 MHz): δ 8.08-8.02 (m, 2H),7.63-7.56 (m, 1H), 7.49-7.42 (m, 2H), 7.21 (q, J=5.2 Hz, 1H), 6.92 (d,J=16.0 Hz, 1H), 6.85 (d, J=16.0 Hz, 1H), 3.81 (s, 3H), 1.69 (d, J=5.2Hz, 3H). MS (ESI): m/z 278.99 (M+H)⁺.

Example 44 Cyclohexylcarbonyloxybutyl methyl(2E)but-2-ene-1,4-dioate(44)

Following general procedure A, methyl hydrogen fumarate (1.00 g, 7.68mmol) was reacted with CsHCO₃ (2.22 g, 11.52 mmol) and chlorobutylcyclohexanecarboxylate (2.16 g, 9.98 mmol) at ca. 55° C. to afford 1.2 g(50% yield) of the title compound (44) as a clear oil after purificationby mass-guided preparatory. HPLC and lyophilization. ¹H NMR (CDCl₃, 400MHz): δ 6.90-6.77 (m, 3H), 3.81 (s, 3H), 2.34-2.28 (m, 1H), 1.91-1.88(m, 2H), 1.82-1.73 (m, 4H), 1.65-1.62 (m, 2H), 1.47-1.39 (m, 4H),1.29-1.23 (m, 2H), 0.98-0.94 (t, 3H). MS (ESI): m/z 313.09 (M+H)⁺.

Example 45 [(2E)-3-(Methoxycarbonyl)prop-2-enoyloxy]ethylmethyl(2E)but-2-ene-1,4-dioate (45)

Adopting methods and procedures according to Iyer, et al., Synth.Commun. 1995, 25(18), 2739, chloroethyl methyl(2E)but-2-ene-1,4-dioatewas prepared from methyl hydrogen fumarate (MHF), acetaldehyde, andanhydrous zinc(II) chloride in anhydrous dichloromethane (DCM). ¹H NMR(CDCl₃, 400 MHz): δ 6.95 (d, J=16.0 Hz, 1H), 6.85 (d, J=16.0 Hz, 1H),6.60 (q, J=5.2 Hz, 1H), 3.81 (s, 3H), 1.83 (d, J=5.2 Hz, 3H).

Following general procedure A, methyl hydrogen fumarate (0.22 g, 1.7mmol) in NMP (4 mL) was reacted with CsHCO₃ (0.38 g, 1.9 mmol) andchloroethyl methyl(2E)but-2-ene-1,4-dioate (0.27 g, 1.4 mmol) to afford0.068 g (17% yield) of the title compound (45) after purification bysilica gel flash chromatography (Biotage) using a mixture of ethylacetate (EtOAc) and hexanes (1:3). ¹H NMR (CDCl₃, 400 MHz): δ 7.03 (q,J=5.2 Hz, 1H), 6.90 (d, J=16.0 Hz, 1H), 6.82 (d, J=16.0 Hz, 1H), 3.82(s, 3H), 1.60 (d, J=5.2 Hz, 3H).

Example 46 Methyl2-methyl-1-phenylcarbonyloxypropyl(2E)but-2-ene-1,4-dioate (46)

Following general procedure A, methyl hydrogen fumarate (0.50 g, 3.82mmol) was reacted with CsHCO₃ (5.76 mmol) and chloroisobutyl benzoate(1.1 g, 5.17 mmol) at ca. 55° C. to afford 0.18 g (15% yield) of thetitle compound (46) after purification by silica gel flashchromatography (Biotage) using a mixture of ethyl acetate (EtOAc) andhexanes (1:7). ¹H NMR (CDCl₃, 400 MHz): δ 8.04-8.01 (m, 2H), 7.58-7.55(m, 1H), 7.45-7.41 (m, 2H) 6.98 (d, J=4.8 Hz, 1H), 6.90 (d, J=16.0 Hz,1H), 6.84 (d, J=16.0 Hz, 1H), 3.78 (s, 3H), 2.25-2.21 (m, 1H), 1.10-1.07(m, 6H); MS (ESI): m/z 307.11 (M+H)⁺.

Example 47 (2E)-3-[(2-Morpholin-4-yl-2-oxoethyl)oxycarbonyl]prop-2-enoicacid (47)

Following general procedure A, fumaric acid (1.00 g, 8.60 mmol) wasreacted with chloro acetyl morpholine (1.4 g, 8.6 mmol) and Ag₂O (4.2 g,9.47 mmol) at ca. 100° C. in N-methylpyrrolidinone. The reaction mixturewas cooled to room temperature, filtered over Celite®, and the filtercake was washed with ethylacetate (EtOAc). The combined organicfiltrates were subjected to acidic aqueous work-up and the crudematerial was purified by mass-guided preparative HPLC to give 0.50 g(24% yield) of the title compound (47) as a white solid. ¹H NMR (CD₃CN,400 MHz): δ 6.85-6.80 (m, 2H), 4.85 (s, 2H), 3.64-3.60 (m, 4H),3.57-3.54 (t, J=4.8 Hz, 2H), 3.42-3.39 (t, J=5.2 Hz, 2H). MS (ESI): m/z244.06 (M+H)⁺, 242.07 (M−H)⁻.

Example 48(2E)-3-{[(N,N-Diethylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic acid (48)

Following general procedure A, mono tert-butyl fumaric acid (0.70 g,4.06 mmol) was reacted with N,N-diethyl chloroacetamide (0.55 mL, 0.60g, 4.06 mmol) and CsHCO₃ (0.93 g, 4.87 mmol) at ca. 55° C. inN-methylpyrrolidinone (NMP). After isolation and purification, the crudematerial was reacted in 50 vol-% trifluoroacetic acid (TFA) indichloromethane (DCM). The free acid was purified by mass-guidedpreparative HPLC to afford 0.051 g (6% yield) of the title compound (48)as a white solid. ¹H NMR (CD₃CN, 400 MHz): δ 6.90-6.82 (m, 2H), 4.88 (s,2H), 3.40-3.32 (q, J=6.8 Hz, 2H), 3.31-3.27 (q, J=6.8 Hz, 2H), 1.22-1.18(t, J=7.6 Hz, 3H), 1.11-1.07 (t, J=6.8 Hz, 3H). MS (ESI): m/z=230.03(M+H)⁺, 228.07 (M−H)⁻.

Example 49 (2E)-3-{[(2-Methylpropanoyloxy)ethyl]oxycarbonyl}prop-2-enoicacid (49)

Following general procedure A, chloroethyl 2-methylpropanoate (0.24 g,1.58 mmol) was reacted overnight with the pre-formedbis-dicyclohexylamine salt (DCHA) of fumaric acid (FA) (0.50 g, 1.26mmol) in N-methylpyrrolidinone (NMP) at ca 100° C. The crude materialwas purified by mass-guided preparative HPLC to afford (0.035 g, 12%yield) of the title compound (49) as a white, waxy, semi-solid. ¹H NMR(CD₃CN, 400 MHz): δ 6.89-6.83 (q, J=5.6 Hz, 1H), 6.82-6.81 (d, J=16.0Hz, 1H), 6.73-6.69 (d, J=16.0 Hz, 1H), 2.58-2.49 (heptet, J=6.8 Hz, 1H),1.49-1.47 (d, J=5.6 Hz, 3H), 1.12 (d, J=7.2 Hz, 3H), 1.09 (d, J=6.8 Hz,6H); MS (ESI): m/z=239.01 (M−H)⁻.

Example 50(2E)-3-({[(Methylethyl)oxycarbonyloxy]ethyl}oxycarbonyl)prop-2-enoicacid (50)

Following general procedure A, chloroethyl (methylethoxy)formate (0.25g, 1.50 mmol) was reacted overnight with the pre-formedbis-dicyclohexylamine salt (DCHA) of fumaric acid (FA) (0.50 g, 1.26mmol) in N-methylpyrrolidinone (NMP) at ca. 100° C. The crude materialwas purified by mass-guided preparative HPLC to afford (0.030 g, 10%yield) of the title compound (50) as a white, waxy, semi-solid. ¹H NMR(CD₃CN, 400 MHz): δ 6.86 (q, J=5.6 Hz, 1H), 6.81 (d, J=16.0 Hz, 1H),6.73 (d, J=16.0 Hz, 1H), 2.57-2.50 (heptet, J=7.2 Hz, 1H), 1.48 (d,J=5.6 Hz, 3H), 1.13 (m, 6H). MS (ESI): m/z=244.99 (M−H)⁻.

Example 51 Alkyloxy- and Aryloxy-carbonyloxyalkyl Alkyl HydrogenFumarates

The following alkyloxy- and aryloxy-carbonyloxyalkyl hydrogen fumarateswere prepared using the methods described in Examples 1-50 and adaptinggeneral synthetic procedures A, B1, and B2:

-   (2E)-3-({[N-benzylcarbamoyl]methyl}oxycarbonyl) prop-2-enoic acid;-   (2E)-3-[(2-morpholin-4-yl-2-oxoethyl) oxycarbonyl]prop-2-enoic acid;-   (2E)-3-{[(N-butylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic acid;-   (2E-3-{[N-methoxy-N-methylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   bis-(2-methoxyethylamino)carbamoyl]methyl prop-2-enoic acid;-   N,N-dimethylcarbamoyl)methyl pro-2-enoic acid;-   2-[(2E)-3-(methoxycarbonyl) prop-2-enoyloxy]acetic acid;-   (2E)-3-({[N-(3-carboxypropyl)carbamoyl]methyl}oxycarbonyl)    prop-2-enoic acid;-   (2E)but-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl prop-2-enoic acid;-   (2E)-3-[(2-{(2S)-2-[tert-butyl)    oxycarbonyl]pyrrolidinyl}-2-oxoethyl) oxycarbonyl]prop-2enoic acid;-   1-[2-((2E)-3-carboxyprop-2-enoyloxy)acetyl](2S)    pyrrolidine-2-carboxylic acid;-   (2E)-3-[([N-{(ethoxycarbonyl)methyl]-N-methylcarbamoyl}methyl)    oxycarbonyl]prop-2-enoic acid;-   (2E)-3-{[(N-{[(tert-butyl)    oxycarbonyl]methyl}-N-methylcarbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-[(1-methyl-2-morpholin-4-yl-2-oxoethyl)    oxycarbonyl]prop-2-enoic acid;-   (2E)-3-({[N,N-bis(2-methoxyethyl)carbamoyl]ethyl}oxycarbonyl)    prop-2-enoic acid;-   (2E)-3-{[(N,N-dimethylcarbamoyl)ethyl]oxycarbonyl}prop-2-enoic acid;-   (2E)-3-[({N,N-bis[2-methylethoxy)ethyl]carbamoyl}methyl)    oxycarbonyl]prop-2-enoic acid;-   (2E)-3-({[N,N-bis(2-ethoxyethyl)carbamoyl]methyl}oxycarbonyl)    prop-2-enoic acid;-   (2E)-3-{[2-(4-acetylpiperazinyl)-2-oxoethyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-({2-oxo-2-[4-benzylpiperazinyl]ethyl}oxycarbonyl)prop-2-enoic    acid;-   (2E)-3-{[(N-{[(tert-butyl)oxycarbonyl]methyl}carbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-{[(N-methyl-N-{[(methylethyl)oxycarbonyl]methyl}carbamoyl)methyl]oxycarbonyl}prop-2-enoic    acid;-   (2E)-3-[({N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methyl)oxycarbonyl]prop-2-enoic    acid;-   (2E)-3-[({N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}ethyl)oxycarbonyl]prop-2-enoic    acid; and-   (2E)-3-[({N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl}ethyl)oxycarbonyl]prop-2-enoic    acid.

Example 52 Methods for Determining Stability of Prodrugs In Vitro

For a prodrug, it can be desirable that the prod rug remains intact(i.e., uncleaved) while in the systemic circulation and be cleaved(i.e., to release the parent drug) in the target tissue. Alternatively,it can be desirable that the prodrug remains intact (i.e., uncleaved)while in the gastrointestinal tract and be cleaved (i.e., to release theparent drug) after being absorbed or taken up from the gastrointestinallumen, e.g., in either the enterocytes lining the gastrointestinal lumenor in the blood. A useful level of stability can at least in part bedetermined by the mechanism and pharmacokinetics of the prodrug. Ingeneral, prodrugs that are more stable in pancreatin or colonic washassay and are more labile in rat plasma, human plasma, rat liver S9,and/or human liver S9 preparations can be useful as an orallyadministered prodrug. In general, prodrugs that are more stable in ratplasma, human plasma, rat liver S9, and/or human liver S9 preparationsand which are more labile in cell homogenate preparations, such CaCo2 S9preparations, can be useful as systemically administered prodrugs and/orcan be more effective in delivering a prodrug to a target tissue. Ingeneral, prodrugs that are more stable in a range of pH physiologicalbuffers (pH 6.0 to pH 8.5) can be more useful as prodrugs. In general,prodrugs that are more labile in cell homogenate preparations, suchCaCo2 S9 preparations, can be intracellularly cleaved to release theparent drug to a target tissue. The results of tests, such as thosedescribed in this example, for determining the enzymatic or chemicalcleavage of prodrugs in vitro can be used to select prodrugs for in vivotesting.

The stabilities of prodrugs can be evaluated in one or more in vitrosystems using a variety of preparations following methods known in theart. For example, methods used to determine the stability of prodrugs inCaco2 S9 homogenate, rat liver S9, rat plasma, porcine pancreatin, ratcolonic wash, and pH 8.0 buffer are described herein.

CaCo2 S9 homogenate was prepared using the following procedure. CaCo2cells were grown in culture for 21 days prior to harvesting. Culturemedium was removed from the culture vessel and the monolayer was rinsedtwice with 10-15 mL chilled PBS buffer. PBS buffer (7-10 mL) was addedto the flask and the cells scraped from the growth surface andtransferred to a centrifuge tube. The cells were pelleted bycentrifugation at 1500 rpm for 5 min at 4° C. The supernatant wasremoved and the cell pellet washed with ice cooled PBS and repelleted bycentrifugation. The supernatant was removed and the pellet resuspendedin cell lysis buffer (0.1SM KCl and 10 mM sodium phosphate buffer, pH7.4). Cells were lysed by sonication at 4° C. using a probe sonicator.The lysed cells were then transferred to vials and centrifuged at 1600rpm for 10 min at 4° C. to remove intact cells, nuclei, and largecellular debris. The supernatant was removed and transferred to a tubefor centrifugation at 8600 rpm for 20 min at 4° C. After centrifugation,the resulting supernatant representing the CaCo2 cell homogenate S9fraction was carefully removed and aliquoted into vials for storage at−80° C. until the time of use. At the time of use, CaCo2 S9 lysate wasdiluted to 0.5 mg/mL in 0.1M Tris buffer, pH 7.4.

Rat liver S9 (XenoTech, Lenexa, Kans.; R1000.S9, 20 mg/mL) was dilutedto 0.5 mg mL in 0.1 M potassium phosphate buffer at pH 7.4 and 1 mMNADPH cofactor.

Rat plasma (Pel-Freez® Biologicals, Rogers, Ark.; 36150) was used asobtained from the supplier.

Porcine pancreatin (Sigma Aldrich, St. Louis, Mo.; P1625-100G) was diledto 10 mg/mL in 0.1M Tris buffer, pH 7.4.

To prepare the rat colonic wash, the colon between the ceacum and rectumwas resected from a euthanized rat. Five to 10 mL of PBS pH 7.4 buffer(depending on the weight of the rat) was flushed into the lumen of thelarge intestine and collected into a 250 mL glass beaker at 0° C. (icebath). The colonic wash was transferred into 10 mL conical tubes using a10 mL syringe fitted with a filter. Samples of 0.5 mL colonic wash arestored at −80° C. until the time of use. Colonic was used withoutdilution.

The enzymatic stability assays for prodrug in CaCo2 S9, rat liver S9,rat plasma, pig pancreatin, and rat colonic was were performed using thefollowing procedure. Ninety (90) μL of lysate is aliquoted to designatedtubes on a cluster plates. The lysate was preincubated for 10 min at 37°C. With the exception of the t(0) time point, 10 μL of a 400 μM solutionof test compound in 0.1M Tris buffer, pH 7.4 was added to multiple wellsrepresenting different incubation times. The samples were incubated at37° C. At each time point, the reaction was quenched by adding 300 μL of100% ethanol. The samples were thoroughly mixed, the tubes transferredto a V-bottom plate, and stored at −20° C. For the t(0) time point, thelysate was quenched with 300 μL of ice cold 100% ethanol, thoroughlymixed, 10 μL of 400 KIM test compound was added and mixed, and thesample tube transferred to a V-bottom plate and stored at −20° C. Foranalysis, 180 μL from each sample was transferred to a 96 well V-bottomplate and sealed. After all time points were collected, the plate wascentrifuged for 10 min at 5600 rpm at 4° C. One-hundred fifty (150) μLfrom each well was then transferred to a 96 well round bottom plate.Samples were analyzed using LC/MS/MS to determine the concentrations ofprodrug and parent drug.

For the pH 8.0 stability studies, 190 μL of 150 mM NaH₂PO₄ buffer pH 8.0was added to each sample tube. Ten (10) μL of 20 mM test compound wasadded to each tube and mixed. The samples were incubated for 60 min at37° C. Following incubation, the samples were transferred to roomtemperature and 800 μL of 50% ACN in water was added to each tube.Samples were analyzed using LC/MS/MS to determine the concentrations ofprodrug and parent drug.

LC-MS/MS analysis for MHF was performed using an API 4000 equipped withan Agilent 1100 HPLC and a Leap Technologies autosampler. A HPLCPhenomenex Onyx Monolithic C18 (CH0-7644) column at a temperature of 35°C., flow rate of 2.0 mL/min, injection volume of 30 μL, and a 3 min runtime was used. Mobile phase AI was 0.1% formic acid in water and Mobilephase AII was 0.1% formic acid in acetonitrile. The gradient was 98%AI/2% AII at time 0; 98% AI/2% AII at time 0.1 min; 5% AI/95% AII attime 1.4 min; 5% AI/95% AII at time 2.2 min; 98% AI/2% AII at time 2.3min; and 98% AI/2% AII at time 3.0 min. MHF content was determined usingnegative ion mode (Q1 128.94; Q2 71).

The stability of DMF and certain MHF prodrugs provided by the presentdisclosure in various media are presented Table 1.

TABLE 1 Stability of MHF prodrugs in biological media. T_(1/2) ParentProdrug Cleavage (min) rColonic Cmpd CaCo2 rLiver rPlasma pPancreatinWash pH 8.0^(§) DMF 2 1 2 0 >60 0 10  3 1 1 4 >60 42 4 5 2 1 2 33 40 2 49 9 1 25 17 9 8 6 1 4 >60 47 1 8 2 1 8 >60 63 5 30 10 2 3 >60 53 3 56 221 8 >60 53 8 52 23 6 15 27 141 ^(§)Percent DMF or prodrug remainingafter 60 minutes.

Example 53 Methyl Hydrogen Fumarate Bioavailability Following OralAdministration of Methyl Hydrogen Fumarate Prodrugs

Rats were obtained commercially and were pre-cannulated in the jugularvein. Animals were conscious at the time of the experiment. All animalswere fasted overnight and until 4 hours post-dosing of a prodrug ofFormula (I).

Rat blood samples (0.3 mL/sample) were collected from all animals priorto dosing and at different time-points up to 24 h post-dose into tubescontaining EDTA. Two aliquots (100 μL each) were quenched with 300 μLmethanol and stored at −20° C. prior to analysis.

To prepare analysis standards, 90 μL of rat blood was quenched with 300μL methanol followed by 10 μL of spiking standard and/or 20 μL ofinternal standard. The sample tubes were vortexed for at least 2 min andthen centrifuged at 3400 rpm for 20 min. The supernatant was thentransferred to an injection vial or plate for analysis by LC-MS-MS.

To prepare samples for analysis, 20 μL of internal standard was added toeach quenched sample tube. The sample tubes were vortexed for at least 2min and then centrifuged at 3400 rpm for 20 min. The supernatant wasthen transferred to an injection vial or plate for analysis by LC-MS-MS.

LC-MS-MS analysis was performed using an API 4000 (MS12) equipped withAgilent 1100 HPLC and a Leap Technologies autosampler. The followingHPLC column conditions were used: HPLC column: Onyx Monolithic C18Phenomex (PN CH0-7644), 35C; flow rate 2.0 mL/min; injection volume 30μL; run time 3 min; mobile phase A: 0.1% formic acid in water; mobilephase B: 0.1% formic acid in acetonitrile (ACN); gradient: 98% A/2% B at0.0 min; 98% A/2% B at 0.1 min; 5% A/95% B at 1.4 min; 5% A/95% B at 2.2min; 98% A/2% B at 2.3 min; and 98% A/2% B at 3.0 min. MHF was monitoredin negative ion mode.

Non-compartmental analysis was performed using WinNonlin software (v.3.1Professional Version, Pharsight Corporation, Mountain View, Calif.) onindividual animal profiles. Summary statistics on major parameterestimates was performed for C_(max) (peak observed concentrationfollowing dosing), T_(max) (time to maximum concentration is the time atwhich the peak concentration was observed), AUC_((0-t)) (area under theplasma concentration-time curve from time zero to last collection time,estimated using the log-linear trapezoidal method), AUC_((0-∞)), (areaunder the plasma concentration time curve from time zero to infinity,estimated using the log-linear trapezoidal method to the last collectiontime with extrapolation to infinity), and t_(1/2,z) (terminalhalf-life).

MHF, DMF or MHF prodrug was administered by oral gavage to groups offour to six adult male Sprague-Dawley rats (about 250 g). Animals wereconscious at the time of the experiment. MHF, DMF or MHF prodrug wasorally or colonically administered in 3.4% Phosal at a dose of 70mg-equivalents MHF per kg body weight.

The percent relative bioavailability (F %) of MHF was determined bycomparing the area under the MHF concentration vs time curve (AUC)following oral or colonic administration of DMF, MHF or MHF prodrug withthe AUC of the MHF concentration vs time curve following intravenousadministration of MHF on a dose normalized basis.

The MHF prodrugs (41), (3), (9), and (11), when administered perorallyto rats at a dose of 30 mg/kg MHF-equivalents in 50 mM sodium acetate pH4.6 exhibited an absolute oral bioavailability (relative to IV) rangingfrom about 43% to about 60% with an average bioavailability of about51%.

Example 54 EAE Animal Model for Assessing Therapeutic Efficacy of MHFProdrugs for Treating Multiple Sclerosis

Animals and EAE Induction

Female C57BL/6 mice, 8-10 weeks old (Harlan Laboratories, Livermore,Calif.), were immunized subcutaneously in the flanks and mid-scapularregion with 200 μg of myelin oligodendrocyte glycoprotein peptide(MOG₃₅₋₅₅) (synthesized by Invitrogen) emulsified (1:1 volume ratio)with complete Freund's adjuvant (CFA) (containing 4 mg/mL Mycobacteriumtuberculosis). Emulsion was prepared by the syringe-extrusion methodwith two glass Luer-Lock syringes connected by a 3-way stopcock. Micewere also given an intraperitoneal injection of 200 ng pertussis toxin(List Biological Laboratories, Inc, Campbell, Calif.) on the day ofimmunization and on day two post immunization. Mice were weighed andexamined daily for clinical signs of experimental autoimmuneencephalomyelitis (EAE). Food and water was provided ad libitum and onceanimals start to show disease, food was provided on the cage bottom. Allexperiments were approved by the Institutional Animal Care and UseCommittee.

Clinical Evaluation

Mice were scored daily beginning on day 7 post immunization. Theclinical scoring scale was as follows (Miller and Karplus, CurrentProtocols in Immunology 2007, 15.1.1-15.1.18): 0=normal; 1=limp tail orhind limb weakness (defined by foot slips between bars of cage top whilewalking); 2=limp tail and hind limb weakness; 3=partial hind limbparalysis (defined as no weight bearing on hind limbs but can still moveone or both hind limbs to some extent); 4=complete hind limb paralysis;5=moribund state (includes forelimb paralysis) or death.

Treatment

DMF or MHF prodrug are dissolved in 0.5% methocellulose/0.1% Tween80 indistilled water and administered by oral gavage twice daily startingfrom day 3 post-immunization until termination. Dexamethasone wasdissolved in 1×PBS buffer and administered subcutaneously once daily.Treatment groups were as follows: vehicle alone, 15 mg/kg DMF, 20 mg/kgMHF prodrug, and 1 mg/kg dexamethasone.

Description 1 Use of an Animal Model to Assess Efficacy in TreatingPsoriasis

The severe, combined immunodeficient (SCID) mouse model can be used toevaluate the efficacy of compounds for treating psoriasis in humans(Boehncke, Ernst Schering Res Found Workshop 2005, 50, 213-34; andBhagavathula et al., J Pharmacol Expt'l Therapeutics 2008, 324(3),938-947).

SCID mice are used as tissue recipients. One biopsy for each normal orpsoriatic volunteer is transplanted onto the dorsal surface of arecipient mouse. Treatment is initiated 1 to 2 weeks aftertransplantation. Animals with the human skin transplants are dividedinto treatment groups. Animals are treated twice daily for 14 days. Atthe end of treatment, animals are photographed and then euthanized. Thetransplanted human tissue along with the surrounding mouse skin issurgically removed and fixed in 10% formalin and samples obtained formicroscopy. Epidermal thickness is measured. Tissue sections are stainedwith an antibody to the proliferation-associated antigen Ki-67 and withan anti-human CD3⁺ monoclonal antibody to detect human T lymphocytes inthe transplanted tissue. Sections are also probed with antibodies toc-myc and β-catenin. A positive response to treatment is reflected by areduction in the average epiderma thickness of the psoriatic skintransplants. A positive response is also associated with reducedexpression of Ki-67 in keratinocytes.

Description 2 Animal Model for Assessing Therapeutic Efficacy of MHFProdrugs for Treating Multiple Sclerosis

Experiments are conducted on female mice aged 4-6 weeks belong to theC57BL/6 strain weighing 17-20 g. Experimental autoimmuneencephalomyelitis (EAE) is actively induced using ≧95% pure syntheticmyelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55,MEVGWYRSPFSRVVHLYRNGK). Each mouse is anesthetized and receives 200 μgof MOG peptide and 15 μg of Saponin extract from Quilija bark emulsifiedin 100 μL of phosphate-buffered saline. A 25 μL volume is injectedsubcutaneously over four flank areas. Mice are also intraperitoneallyinjected with 200 ng of pertussis toxin in 200 μL of PBS. A second,identical injection of pertussis toxin is given after 48 h.

A MHF prodrug is administered at varying doses. Control animals receive25 μL of DMSO. Daily treatment extends from day 26 to day 36post-immunization. Clinical scores are obtained daily from day 0post-immunization until day 60. Clinical signs are scored using thefollowing protocol: 0, no detectable signs; 0.5, distal tail limpness,hunched appearance and quiet demeanor; 1, completely limp tail; 1.5,limp tail and hindlimb weakness (unsteady gait and poor grip withhindlimbs); 2, unilateral partial hindlimb paralysis; 2.5, bilateralhindlimb paralysis; 3, complete bilateral hindlimb paralysis; 3.5,complete hindlimb paralysis and unilateral forelimb paralysis; 4, totalparalysis of hindlimbs and forelimbs (Eugster et al., Eur J Immunol2001, 31, 2302-2312).

Inflammation and demyelination are assessed by histology on sectionsfrom the CNS of EAE mice. Mice are sacrificed after 30 or 60 days andwhole spinal cords are removed and placed in 0.32 M sucrose solution at4° C. overnight. Tissues are prepared and sectioned. Luxol fast bluestain is used to observe areas of demyelination. Haematoxylin and eosinstaining is used to highlight areas of inflammation by darkly stainingthe nuclei of mononuclear cells. Immune cells stained with H&E arecounted in a blinded manner under a light microscope. Sections areseparated into gray and white matter and each sector is counted manuallybefore being combined to give a total for the section. T cells areimmunolabeled with anti-CD3+ monoclonal antibody. After washing,sections are incubated with goat anti-rat HRP secondary antibody.Sections are then washed and counterstained with methyl green.Splenocytes isolated from mice at 30 and 60 days post-immunization aretreated with lysis buffer to remove red blood cells. Cells are thenresuspended in PBS and counted. Cells at a density of about 3×10⁶cells/mL are incubated overnight with 20 μg/mL of MOG peptide.Supernatants from stimulated cells are assayed for IFN-γ protein levelsusing an appropriate mouse IFN-γ immunoassay system.

Description 3 Use of an Animal Model to Assess Efficacy in TreatingInflammatory Bowel Disease

Animal models of inflammatory bowel disease are described by Jurjus etal., J Pharmaocol Toxicol Methods 2004, 50, 81-92; Villegas et al.,Int'l Immunopharmacol 2003, 3, 1731-1741; and Murakami et al.,Biochemical Pharmacol 2003, 66, 1253-1261. For example, the followingprotocol can be used to assess the efficacy of a compound for treatinginflammatory bowel disease.

Female ICR mice are used. Mice are divided into treatment groups. Groupsare given either water (control), 5% DSS in tap water is given at thebeginning of the experiment to induce colitis, or various concentrationsof test compound. After administering test compound for 1 week, 5% DSSin tap water is also administered to the groups receiving test compoundfor 1 week. At the end of the experiment, all mice are killed and thelarge intestine is removed. Colonic mucosa samples are obtained andhomogenized. Proinflammatory mediators (e.g., IL-1α, IL-1β, TNF-α, PGE2,and PGF2α) and protein concentrations are quantified. Each excised largeintestine is histologically examined and the damage to the colon scored.

Description 4 Clinical Trial for Assessing Efficacy in Treating Asthma

Adult subjects (nonsmokers) with stable mild-to-moderate asthma areenrolled (see, e.g., Van Schoor and Pauwels, Eur Respir J 2002, 19,997-1002). A randomized, double-blind, placebo-controlled, two-periodcrossover design is used. On screening day 1, patients undergo amethacholine challenge (<8 mg/mL). The baseline forced expiratory volumein one second (FEV1) prior to each subsequent challenge must be within15% of the screening baseline FEV1 obtained at the first visit. Aneurokinin challenge (1×10⁻⁶ mol/mL) on screening day 2 is performed24-72 h later. Study-period one commences within 10 days after visittwo. First, a methacholine and a neurokinin-A (NKA) challenge isperformed on days 1 and 0, respectively. At visit four, test compound isadministered at an appropriate dose and for an appropriate period oftime. On the last 2 days of the treatment period, methacholine and NKAchallenges are repeated. Following treatment-period one, there is awashout period of about 5 weeks, following which the patients crossedover to another medication or placebo in study period two, which isidentical to period one. Pulmonary function tests are performed using aspirometer. The metacholine challenge is performed by inhaling doublingconcentrations of methacholine until the FEV1 falls by >20% of thepostdiluent baseline FEV1 of that day as described by Cockcroft et al.,Clin Allergy 1977, 7, 235-243. NKA challenge is performed by inhalingincreasing concentrations of NKA as described by Van Schoor et al., EurRespir J 1998, 12, 17-23. The effect of a treatment on airwayresponsiveness is determined using appropriate statistical methods.

Description 5 Use of an Animal Model to Assess Efficacy in TreatingChronic Obstructive Pulmonary Disease

An animal model using mice chronically exposed to cigarette smoke can beused for assessing efficacy in treating emphysema (see, e.g., Martoranaet al., Am J Respir Crit. Care Med 2005, 172, 848-835; and Cavarra etal., Am J Respir Crit. Care Med 2001, 164, 886-890). Six-week oldC57B1/6J male mice are used. In the acute study, the mice are exposedeither to room air or to the smoke of five cigarettes for 20 minutes. Inthe chronic study, the mice are exposed to either room air or to thesmoke of three cigarettes/day for 5 days/week for 7 months.

For the acute study, mice are divided into three groups of 40 animalseach. These groups are then divided into four subgroups of 10 mice eachas follows: (1) no treatment/air-exposed; (2) notreatment/smoke-exposed; (3) a first dose of test compound plussmoke-exposed; and (4) a second dose of test compound. In the firstgroup, trolox equivalent antioxidant capacity is assessed at the end ofthe exposure in bronchoalveolar lavage fluid. In the second group,cytokines and chemokines are determined in bronchoalveolar lavage fluidusing a commercial cytokine panel at 4 hours; and in the third groupbronchoalveolar lavage fluid cell count is assessed at 24 hours.

For the chronic study, five groups of animals are used: (1) notreatment/air-exposed; (2) a first dose of a test compound plusair-exposed; (3) no treatment/smoke-exposed; (4) a second dose of thetest compound plus smoke-exposed; and (5) the first dose of the testcompound plus smoke exposed. Seven months after chronic exposure to roomair or cigarette smoke, 5 to 12 animals from each group are killed anthe lungs fixed intratracheally with formalin. Lung volume is measuredby water displacement. Lungs are stained. Assessment of emphysemaincludes mean linear intercept and internal surface area. The volumedensity of macrophages, marked immunohistochemically with antimouseMac-3 monoclonal antibodies is determined by point counting. A mouse isconsidered to have goblet cell metaplasia when at least one or moremidsize bronchi/lung showed a positive periodic acid-Schiff staining.For the determination of desmosine, fresh lungs are homogenized,processed, and analyzed by high-pressure liquid chromatography.

Description 6 Animal Models for Assessing Therapeutic Efficacy of MHFProdrugs for Treating Parkinson's Disease

MPTP Induced Neurotoxicity

MPTP, or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is a neurotoxinthat produces a Parkinsonian syndrome in both man and experimentalanimals. Studies of the mechanism of MPTP neurotoxicity show that itinvolves the generation of a major metabolite, MPP⁺, formed by theactivity of monoamine oxidase on MPTP. Inhibitors of monoamine oxidaseblock the neurotoxicity of MPTP in both mice and primates. Thespecificity of the neurotoxic effects of MPP⁺ for dopaminergic neuronsappears to be due to the uptake of MPP⁺ by the synaptic dopaminetransporter. Blockers of this transporter prevent MPP⁺ neurotoxicity.MPP⁺ has been shown to be a relatively specific inhibitor ofmitochondrial complex I activity, binding to complex I at the retenonebinding site and impairing oxidative phosphorylation. In vivo studieshave shown that MPTP can deplete striatal ATP concentrations in mice. Ithas been demonstrated that MPP⁺ administered intrastriatally to ratsproduces significant depletion of ATP as well as increased lactateconcentration confined to the striatum at the site of the injections.Compounds that enhance ATP production can protect against MPTP toxicityin mice.

A prodrug of Formulae (I)-(IV) is administered to animals such as miceor rats for three weeks before treatment with MPTP. MPTP is administeredat an appropriate dose, dosing interval, and mode of administration for1 week before sacrifice. Control groups receive either normal saline orMPTP hydrochloride alone. Following sacrifice the two striate arerapidly dissected and placed in chilled 0.1 M perchloric acid. Tissue issubsequently sonicated and aliquots analyzed for protein content using afluorometer assay. Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), andhomovanillic acid (HVA) are also quantified. Concentrations of dopamineand metabolites are expressed as nmol/mg protein.

Prodrugs of Formulae (I)-(IV) that protect against DOPAC depletioninduced by MPTP, HVA, and/or dopamine depletion are neuroprotective andtherefore can be useful for the treatment of Parkinson's disease.

Haloperidol-Induced Hypolocomotion

The ability of a compound to reverse the behavioral depressant effectsof dopamine antagonists such as haloperidol, in rodents and isconsidered a valid method for screening drugs with potentialantiparkinsonian effects (Mandhane, et al., Eur. J. Pharmacol 1997, 328,135-141). Hence, the ability of prodrugs of Formulae (I)-(IV) to blockhaloperidol-induced deficits in locomotor activity in mice can be usedto assess both in vivo and potential anti-Parkinsonian efficacy.

Mice used in the experiments are housed in a controlled environment andallowed to acclimatize before experimental use. One and one-half (1.5)hours before testing, mice are administered 0.2 mg/kg haloperidol, adose that reduces baseline locomotor activity by at least 50%. A testcompound is administered 5-60 min prior to testing. The animals are thenplaced individually into clean, clear polycarbonate cages with a flatperforated lid. Horizontal locomotor activity is determined by placingthe cages within a frame containing a 3×6 array of photocells interfacedto a computer to tabulate beam interrupts. Mice are left undisturbed toexplore for 1 h, and the number of beam interruptions made during thisperiod serves as an indicator of locomotor activity, which is comparedwith data for control animals for statistically significant differences.

6-Hydroxydopamine Animal Model

The neurochemical deficits seen in Parkinson's disease can be reproducedby local injection of the dopaminergic neurotoxin, 6-hydroxydopamine(6-OHDA) into brain regions containing either the cell bodies or axonalfibers of the nigrostriatal neurons. By unilaterally lesioning thenigrostriatal pathway on only one-side of the brain, a behavioralasymmetry in movement inhibition is observed. Althoughunilaterally-lesioned animals are still mobile and capable of selfmaintenance, the remaining dopamine-sensitive neurons on the lesionedside become supersensitive to stimulation. This is demonstrated by theobservation that following systemic administration of dopamine agonists,such as apomorphine, animals show a pronounced rotation in a directioncontralateral to the side of lesioning. The ability of compounds toinduce contralateral rotations in 6-OHDA lesioned rats has been shown tobe a sensitive model to predict drug efficacy in the treatment ofParkinson's disease.

Male Sprague-Dawley rats are housed in a controlled environment andallowed to acclimatize before experimental use. Fifteen minutes prior tosurgery, animals are given an intraperitoneal injection of thenoradrenergic uptake inhibitor desipramine (25 mg/kg) to prevent damageto nondopamine neurons. Animals are then placed in an anesthetic chamberand anesthetized using a mixture of oxygen and isoflurane. Onceunconscious, the animals are transferred to a stereotaxic frame, whereanesthesia is maintained through a mask. The top of the head is shavedand sterilized using an iodine solution. Once dry, a 2 cm long incisionis made along the midline of the scalp and the skin retracted andclipped back to expose the skull. A small hole is then drilled throughthe skull above the injection site. In order to lesion the nigrostriatalpathway, the injection cannula is slowly lowered to position above theright medial forebrain bundle at −3.2 mm anterior posterior, −1.5 mmmedial lateral from the bregma, and to a depth of 7.2 mm below theduramater. Two minutes after lowering the cannula, 6-OHDA is infused ata rate of 0.5 μL/min over 4 min, to provide a final dose of 8 μg. Thecannula is left in place for an additional 5 min to facilitate diffusionbefore being slowly withdrawn. The skin is then sutured shut, the animalremoved from the sterereotaxic frame, and returned to its housing. Therats are allowed to recover from surgery for two weeks before behavioraltesting.

Rotational behavior is measured using a rotameter system havingstainless steel bowls (45 cm dia×15 cm high) enclosed in a transparentPlexiglas cover around the edge of the bowl and extending to a height of29 cm. To assess rotation, rats are placed in a cloth jacket attached toa spring tether connected to an optical rotameter positioned above thebowl, which assesses movement to the left or right either as partial(45°) or full (360°) rotations.

To reduce stress during administration of a test compound, rats areinitially habituated to the apparatus for 15 min on four consecutivedays. On the test day, rats are given a test compound, e.g., a prodrugof Formulae (I)-(IV). Immediately prior to testing, animals are given asubcutaneous injection of a subthreshold dose of apomorphine, and thenplaced in the harness and the number of rotations recorded for one hour.The total number of full contralatral rotations during the hour testperiod serves as an index of antiparkinsonian drug efficacy.

Description 7 Animal Model for Assessing Therapeutic Efficacy of MHFProdrugs for Treating Alzheimer's Disease

Heterozygous transgenic mice expressing the Swedish AD mutant gene,hAPPK670N, M671L (Tg2576; Hsiao, Learning & Memory 2001, 8, 301-308) areused as an animal model of Alzheimer's disease. Animals are housed understandard conditions with a 12:12 light/dark cycle and food and wateravailable ad libitum. Beginning at 9 months of age, mice are dividedinto two groups. The first two groups of animals receive increasingdoses of a MHF prodrug, over six weeks. The remaining control groupreceives daily saline injections for six weeks.

Behavioral testing is performed at each drug dose using the samesequence over two weeks in all experimental groups: (1) spatial reversallearning, (2) locomotion, (3) fear conditioning, and (4) shocksensitivity.

Acquisition of the spatial learning paradigm and reversal learning aretested during the first five days of test compound administration usinga water T-maze as described in Bardgett et al., Brain Res Bull 2003, 60,131-142. Mice are habituated to the water T-maze during days 1-3, andtask acquisition begins on day 4. On day 4, mice are trained to find theescape platform in one choice arm of the maze until 6 to 8 correctchoices are made on consecutive trails. The reversal learning phase isthen conducted on day 5. During the reversal learning phase, mice aretrained to find the escape platform in the choice arm opposite from thelocation of the escape platform on day 4. The same performance criteriaand inter-trial interval are used as during task acquisition.

Large ambulatory movements are assessed to determine that the results ofthe spatial reversal learning paradigm are not influenced by thecapacity for ambulation. After a rest period of two days, horizontalambulatory movements, excluding vertical and fine motor movements, areassessed in a chamber equipped with a grid of motion-sensitive detectorson day 8. The number of movements accompanied by simultaneous blockingand unblocking of a detector in the horizontal dimension are measuredduring a one-hour period.

The capacity of an animal for contextual and cued memory is tested usinga fear conditioning paradigm beginning on day 9. Testing takes place ina chamber that contains a piece of absorbent cotton soaked in anodor-emitting solution such as mint extract placed below the grid floor.A 5-min, 3 trial 80 db, 2800 Hz tone-foot shock sequence is administeredto train the animals on day 9. On day 10, memory for context is testedby returning each mouse to the chamber without exposure to the tone andfoot shock, and recording the presence or absence of freezing behaviorevery 10 seconds for 8 minutes. Freezing is defined as no movement, suchas ambulation, sniffing or stereotypy, other than respiration.

On day 11, the response of the animal to an alternate context and to theauditory cue is tested. Coconut extract is placed in a cup and the 80 dBtone is presented, but no foot shock is delivered. The presence orabsence of freezing in response to the alternate context is thendetermined during the first 2 minutes of the trial. The tone is thenpresented continuously for the remaining 8 minutes of the trial, and thepresence or absence of freezing in response to the tone is determined.

On day 12, the animals are tested to assess their sensitivity to theconditioning stimulus, i.e., foot shock.

Following the last day of behavioral testing, animals are anesthetizedand the brains removed, post-fixed overnight, and sections cut throughthe hippocampus. The sections are stained to image β-amyloid plaques.

Data is analyzed using appropriate statistical methods.

Description 8 Animal Model for Assessing Therapeutic Efficacy of MHFProdrugs for Treating Huntington's Disease

Neuroprotective Effects in a Transgenic Mouse Model of Huntington'sDisease

Transgenic HD mice of the N171-82Q strain and non-transgenic littermatesare treated with a prodrug of Formulae (I)-(IV) or a vehicle from 10weeks of age. The mice are placed on a rotating rod (“rotarod”). Thelength of time at which a mouse falls from the rotarod is recorded as ameasure of motor coordination. The total distance traveled by a mouse isalso recorded as a measure of overall locomotion. Mice administeredprodrugs of Formulae (I)-(IV) that are neuroprotective in the N171-82Qtransgenic HD mouse model remain on the rotarod for a longer period oftime and travel farther than mice administered vehicle.

Malonate Model of Huntington's Disease

A series of reversible and irreversible inhibitors of enzymes involvedin energy generating pathways has been used to generate animal modelsfor neurodegenerative diseases such as Parkinson's and Huntington'sdiseases. In particular, inhibitors of succinate dehydrogenase, anenzyme that impacts cellular energy homeostasis, has been used togenerate a model for Huntington's disease.

To evaluate the effect of MHF prodrugs of Formulae (I)-(IV) in thismalonate model for Huntington's disease, a prodrug of Formulae (I)-(IV)is administered at an appropriate dose, dosing interval, and route, tomale Sprague-Dawley rats. A prodrug is administered for two weeks priorto the administration of malonate and then for an additional week priorto sacrifice. Malonate is dissolved in distilled deionized water and thepH adjusted to 7.4 with 0.1 M HCl. Intrastriatal injections of 1.5 μL of3 μmol malonate are made into the left striatum at the level of theBregma 2.4 mm lateral to the midline and 4.5 mm ventral to the dura.Animals are sacrificed at 7 days by decapitation and the brains quicklyremoved and placed in ice cold 0.9% saline solution. Brains aresectioned at 2 mm intervals in a brain mold. Slices are then placedposterior side down in 2% 2,3,5-tiphenyltetrazolium chloride. Slices arestained in the dark at room temperature for 30 min and then removed andplaced in 4% paraformaldehyde pH 7.3. Lesions, noted by pale staining,are evaluated on the posterior surface of each section. The measurementsare validated by comparison with measurements obtained on adjacent Nisslstain sections. Compounds exhibiting a neuroprotective effect andtherefore potentially useful in treating Huntington's disease show areduction in malonate-induced lesions.

Description 9 Animal Model for Assessing Therapeutic Efficacy of MHFProdrugs for Treating Amyotrophic Lateral Sclerosis

A murine model of SOD1 mutation-associated ALS has been developed inwhich mice express the human superoxide dismutase (SOD) mutationglycine-alanine at residue 93 (SOD1). These SOD1 mice exhibit a dominantgain of the adverse property of SOD, and develop motor neurondegeneration and dysfunction similar to that of human ALS. The SOD1transgenic mice show signs of posterior limb weakness at about 3 monthsof age and die at 4 months. Features common to human ALS includeastrocytosis, microgliosis, oxidative stress, increased levels ofcyclooxygenase/prostaglandin, and, as the disease progresses, profoundmotor neuron loss.

Studies are performed on transgenic mice overexpressing human Cu/Zn-SODG93A mutations (B6SJL-TgN (SOD1-G93A) 1 Gur) and non-transgenic B6/SJLmice and their wild litter mates. Mice are housed on a 12-hr day/lightcycle and (beginning at 45 d of age) allowed ad libitum access to eithertest compound-supplemented chow, or, as a control, regular formula coldpress chow processed into identical pellets. Genotyping can be conductedat 21 days of age as described in Gurney et al., Science 1994,264(5166), 1772-1775. The SOD1 mice are separated into groups andtreated with a test compound, e.g., an MHF prodrug, or serve ascontrols.

The mice are observed daily and weighed weekly. To assess health statusmice are weighed weekly and examined for changes inlacrimation/salivation, palpebral closure, ear twitch and pupillaryresponses, whisker orienting, postural and righting reflexes and overallbody condition score. A general pathological examination is conducted atthe time of sacrifice.

Motor coordination performance of the animals can be assessed by one ormore methods known to those skilled in the art. For example, motorcoordination can be assessed using a neurological scoring method. Inneurological scoring, the neurological score of each limb is monitoredand recorded according to a defined 4-point scale: 0—normal reflex onthe hind limbs (animal will splay its hind limbs when lifted by itstail); 1—abnormal reflex of hind limbs (lack of splaying of hind limbsweight animal is lifted by the tail); 2—abnormal reflex of limbs andevidence of paralysis; 3—lack of reflex and complete paralysis; and4—inability to right when placed on the side in 30 seconds or founddead. The primary end point is survival with secondary end points ofneurological score and body weight. Neurological score observations andbody weight are made and recorded five days per week. Data analysis isperformed using appropriate statistical methods.

The rotarod test evaluates the ability of an animal to stay on arotating dowel allowing evaluation of motor coordination andproprioceptive sensitivity. The apparatus is a 3 cm diameter automatedrod turning at, for example, 12 rounds per min. The rotarod testmeasures how long the mouse can maintain itself on the rod withoutfalling. The test can be stopped after an arbitrary limit of 120 sec.Should the animal fall down before 120 sec, the performance is recordedand two additional trials are performed. The mean time of 3 trials iscalculated. A motor deficit is indicated by a decrease of walking time.

In the grid test, mice are placed on a grid (length: 37 cm, width: 10.5cm, mesh size: 1×1 cm 2) situated above a plane support. The number oftimes the mice put their paws through the grid is counted and serves asa measure for motor coordination.

The hanging test evaluates the ability of an animal to hang on a wire.The apparatus is a wire stretched horizontally 40 cm above a table. Theanimal is attached to the wire by its forepaws. The time needed by theanimal to catch the string with its hind paws is recorded (60 sec max)during three consecutive trials.

Electrophysiological measurements (EMG) can also be used to assess motoractivity condition. Electromyographic recordings are performed using anelectromyography apparatus. During EMG monitoring mice are anesthetized.The measured parameters are the amplitude and the latency of thecompound muscle action potential (CMAP). CMAP is measured ingastrocnemius muscle after stimulation of the sciatic nerve. A referenceelectrode is inserted near the Achilles tendon and an active needleplaced at the base of the tail. A ground needle is inserted on the lowerback of the mice. The sciatic nerve is stimulated with a single 0.2 msecpulse at supramaximal intensity (12.9 mA). The amplitude (mV) and thelatency of the response (ms) are measured. The amplitude is indicativeof the number of active motor units, while distal latency reflects motornerve conduction velocity.

The efficacy of test compounds can also be evaluated using biomarkeranalysis. To assess the regulation of protein biomarkers in SOD1 miceduring the onset of motor impairment, samples of lumbar spinal cord(protein extracts) are applied to ProteinChip Arrays with varyingsurface chemical/biochemical properties and analyzed, for example, bysurface enhanced laser desorption ionization time of flight massspectrometry. Then, using integrated protein mass profile analysismethods, data is used to compare protein expression profiles of thevarious treatment groups. Analysis can be performed using appropriatestatistical methods.

Finally, it should be noted that there are alternative ways ofimplementing the embodiments disclosed herein. Accordingly, the presentembodiments are to be considered as illustrative and not restrictive,and the claims are not to be limited to the details given herein, butmay be modified within the scope and equivalents thereof.

What is claimed is:
 1. A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R¹ and R² areindependently chosen from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl; R³ and R⁴ are independently chosen from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl,C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂ cycloalkylalkyl, C₇₋₁₂arylalkyl, and substituted C₇₋₁₂ arylalkyl; or R³ and R⁴ together withthe nitrogen to which they are bonded form a ring chosen from a C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₅₋₁₀ heterocycloalkyl, andsubstituted C₅₋₁₀ heterocycloalkyl; and R⁵ is methyl; wherein eachsubstituent group is independently chosen from halogen, —OH, —CN, —CF₃,═O, —NO₂, benzyl, —C(O)NR¹¹ ₂, —R¹¹, —OR¹¹, —C(O)R¹¹, —COOR¹¹, and —NR¹¹₂ wherein each R¹¹ is independently chosen from hydrogen and C₁₋₄ alkyl.2. The compound of claim 1, wherein each of R¹ and R² is hydrogen. 3.The compound of claim 1, wherein one of R¹ and R² is hydrogen and theother of R¹ and R² is chosen from methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, and sec-butyl.
 4. The compound of claim 1, wherein R³and R⁴ are independently chosen from hydrogen and C₁₋₆ alkyl.
 5. Thecompound of claim 1, wherein R³ and R⁴ together with the nitrogen towhich they are bonded form a C₅₋₁₀ heterocycloalkyl ring.
 6. Thecompound of claim 1, wherein one of R¹ and R² is hydrogen and the otherof R¹ and R² is chosen from hydrogen and C₁₋₆ alkyl; and R³ and R⁴together with the nitrogen to which they are bonded form a ring chosenfrom morpholine, piperazine, and N-substituted piperazine.
 7. Thecompound of claim 1, wherein one of R¹ and R² is hydrogen; and the otherof R¹ and R² is chosen from hydrogen and C₁₋₆ alkyl; R³ is hydrogen; andR⁴ is chosen from hydrogen, C₁₋₆ alkyl, and benzyl.
 8. The compound ofclaim 1, wherein the compound is chosen from:(N,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;methyl[N-benzylcarbamoyl]methyl(2E)but-2-ene-1,4-dioate; methyl2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate;(N-butylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;[N-(2-methoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate;2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}acetic acid;4-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid;methyl(N-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl(2E)but-2ene-1,4-dioate;(N,N-dimethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;(N-methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate;[bis-(2-methoxyethylamino)carbamoyl]methylmethyl(2E)but-2-ene-1,4-dioate; [N-(methoxycarbonyl)carbamoyl]methylmethyl(2E)but-gene-1,4-dioate; methyl2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate; methyl2-oxo-2-(2-oxo(1,3-oxazolidin-3-yl)ethyl(2E)but-2ene-1,4-dioate;{N[2-(dimethylamino)ethyl]carbamoyl}methyl methyl(2E)but-2ene-1,4dioate; methyl2-(4-methylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate; methyl{N-[(propylamino)carbonyl]carbamoyl}methyl(2E)but-2ene-1,4-dioate;2-(4-acetylpiperazinyl)-2-oxoethyl methyl(2E)but-2ene-1,4-dioate;{N,N-bis[2-(methylethoxy)ethyl]carbamoyl}methylmethyl(2E)but-2-ene-1,4-dioate; methyl2-(4-benzylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate;[N,N-bis(2-ethoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate;2-{(2S)-2-[(tert-butyl)oxycarbonyl]pyrrolidinyl}-2-oxoethylmethyl(2E)but-2ene-1,4-dioate;1-[2-{(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetyl}(2S)pyrrolidine-2-carboxylicacid; (N-1 {[tert-butyl)oxycarbonyl]methyl}-N-methylcarbamoyl)methylmethyl(2E)but-2ene-1,4-dioate;{N-(ethoxycarbonyl)methyl]-N-methylcarbamoyl}methylmethyl(2E)but-2-ene-1,4-dioate; methyl1-methyl-2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate;[N,N-bis(2-methoxyethyl)carbamoyl]ethyl methyl(2E)but-2-ene-1,4-dioate;(N,N-dimethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate;2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxyl]-N-methylacetylamino]acetic acid;(N-{[(tert-butyl)oxycarbonyl]methyl}carbamoyl)methylmethyl(2E)but-2-ene-1,4-dioate;methyl(N-methyl-N-{[(methylethyl)oxycarbonyl]methyl}carbamoyl)methyl(2E)but-2-ene-1,4-dioate;{N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methylmethyl(2E)but-2-ene-1,4-dioate;1N-[ethoxycarbonyl)methyl]-N-benzylcarbamoyl}ethylmethyl(2E)but-2-ene-1,4-dioate;{N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl}ethylmethyl(2E)but-2-ene-1,4-dioate;(1S)-1-methyl-2-morpholin-4-yl-2-oxoethylmethyl(2E)but-2-ene-1,4-dioate; (1S)-1-[N,N-bis(2-methoxyethyl)carbamoyl]ethyl methyl(2E)but-2-ene-1,4-dioate;(1R)-1-(N,N-diethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate;(N-[(methoxycarbonyl)ethyl]carbamoyl)methylmethyl(2E)but-2-ene-1,4-dioate;2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}propanoicacid; and a pharmaceutically acceptable salt of any of the foregoing. 9.A pharmaceutical composition comprising a compound of claim 1 and atleast one pharmaceutically acceptable vehicle.
 10. The pharmaceuticalcomposition of claim 9, which is an oral formulation.
 11. The compoundof claim 1, wherein R³ and R⁴ are independently chosen from methyl,ethyl, methoxyethyl; or R³ and R⁴ together with the nitrogen to whichthey are bonded form a 4-morpholinyl group.
 12. The compound(N,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate.
 13. Thecompound methyl 2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate. 14.The compound bis-(2-methoxyethylamino)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate.
 15. The compound (N,N-dimethylcarbamoyl)methylmethyl(2E)but-2-ene-1,4-dioate.
 16. A pharmaceutical compositioncomprising a compound of any of claims 11 to 15 and at least onepharmaceutically acceptable vehicle.
 17. The pharmaceutical compositionof claim 16, which is an oral formulation.
 18. The pharmaceuticalcomposition of claim 16, which is an oral sustained release formulation.19. The pharmaceutical composition of claim 16, which is an oralcontrolled release formulation.
 20. The pharmaceutical composition ofclaim 16, comprising a second therapeutic agent.
 21. The pharmaceuticalcomposition of claim 16, comprising a second therapeutic agent anantihistamine, a selective serotonin reuptake inhibitor (SSRI), anorepinephrine and serotonin reuptake inhibitor (NSRI), a salicylateselected from a non-steroidal anti-inflammatory agent and a cox-2inhibitor.
 22. The pharmaceutical composition of claim 16, comprising asecond therapeutic agent selected from vitamin E, paroxetine, gabapentinand pentoxifylline.